Stability and Conformation of a Chemoreceptor HAMP Domain Chimera Correlates with Signaling Properties.

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.

Bacterial Energy Sensor Aer Modulates the Activity of the Chemotaxis Kinase CheA Based on the Redox State of the Flavin Cofactor.

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.

Structure of the frequency-interacting RNA helicase: a protein interaction hub for the circadian clock.

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.

Architecture of the soluble receptor Aer2 indicates an in-line mechanism for PAS and HAMP domain signaling.

Bacterial receptors typically contain modular architectures with distinct functional domains that combine to send signals in response to stimuli. Although the properties of individual components have been investigated in many contexts, there is little information about how diverse sets of modules work together in full-length receptors. Here, we investigate the architecture of Aer2, a soluble gas-sensing receptor that has emerged as a model for PAS (Per-Arnt-Sim) and poly-HAMP (histidine kinase-adenylyl cyclase-methyl-accepting chemotaxis protein-phosphatase) domain signaling. The crystal structure of the heme-binding PAS domain in the ferric, ligand-free form, in comparison to the previously determined cyanide-bound state, identifies conformational changes induced by ligand binding that are likely essential for the signaling mechanism. Heme-pocket alternations share some similarities with the heme-based PAS sensors FixL and EcDOS but propagate to the Iß strand in a manner predicted to alter PAS-PAS associations and the downstream HAMP junction within full-length Aer2. Small-angle X-ray scattering of PAS and poly-HAMP domain fragments of increasing complexity allow unambiguous domain assignments and reveal a linear quaternary structure. The Aer2 PAS dimeric crystal structure fits well within ab initio small-angle X-ray scattering molecular envelopes, and pulsed dipolar ESR measurements of inter-PAS distances confirm the crystallographic PAS arrangement within Aer2. Spectroscopic and pull-down assays fail to detect direct interactions between the PAS and HAMP domains. Overall, the Aer2 signaling mechanism differs from the Escherichia coli Aer paradigm, where side-on PAS-HAMP contacts are key. We propose an in-line model for Aer2 signaling, where ligand binding induces alterations in PAS domain structure and subunit association that is relayed through the poly-HAMP junction to downstream domains.

Structure of full-length Drosophila cryptochrome.

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.

Endogenous nitric oxide regulates the recovery of the radiation-resistant bacterium Deinococcus radiodurans from exposure to UV light.

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.

Conformational switching in the fungal light sensor Vivid.

The Neurospora crassa photoreceptor Vivid tunes blue-light responses and modulates gating of the circadian clock. Crystal structures of dark-state and light-state Vivid reveal a light, oxygen, or voltage Per-Arnt-Sim domain with an unusual N-terminal cap region and a loop insertion that accommodates the flavin cofactor. Photoinduced formation of a cystein-flavin adduct drives flavin protonation to induce an N-terminal conformational change. A cysteine-to-serine substitution remote from the flavin adenine dinucleotide binding site decouples conformational switching from the flavin photocycle and prevents Vivid from sending signals in Neurospora. Key elements of this activation mechanism are conserved by other photosensors such as White Collar-1, ZEITLUPE, ENVOY, and flavin-binding, kelch repeat, F-BOX 1 (FKF1).

Structure of Plasmodium falciparum dihydroorotate dehydrogenase with a bound inhibitor.

Membrane-associated dihydroorotate dehydrogenase (DHODH) is an antimalarial therapeutic target without an effective inhibitor. Studies on human DHODH (HsDHODH) led to a structural mechanistic model in which respiratory quinones bind in a tunnel formed by the highly variable N-terminus that leads to the flavin mononucleotide-binding site. The therapeutic agents leflunomide (Arava) and brequinar sodium inhibit HsDHODH by binding in this tunnel. Plasmodium falciparum DHODH (PfDHODH) and HsDHODH have markedly different sensitivities to the two drugs. To understand the structural basis of this differential sensitivity and begin a structure-based drug-design cycle for PfDHODH inhibitors, the three-dimensional structure (2.4 Angstroms, R = 20.1%) of PfDHODH bound to the active metabolite of leflunomide was determined by X-ray crystallography. Comparison of the structures of HsDHODH and PfDHODH reveals a completely different binding mode for the same inhibitor in these two catalytically identical enzymes and explains the previously observed species-specific preferential binding. Because no effective inhibitors have been described for PfDHODH, this structure provides critical insight for the design of potential antimalarials.

Human methionine aminopeptidase type 2 in complex with L- and D-methionine.

Human methionine aminopeptidase type 2 (hMetAP-2) was identified as the molecular target of anti-angiogenic agents such as fumagillin and its analogues. We describe here the crystal structure of hMetAP-2 in complex with l-methionine and d-methionine at 1.9 and 2.0A resolution, respectively. The comparison of the structure of the two complexes establishes the basis of enantiomer discrimination and provides some considerations for the design of selective MetAP-2 inhibitors.

Nitration of a peptide phytotoxin by bacterial nitric oxide synthase.

Nitric oxide (NO) is a potent intercellular signal in mammals that mediates key aspects of blood pressure, hormone release, nerve transmission and the immune response of higher organisms. Proteins homologous to full-length mammalian nitric oxide synthases (NOSs) are found in lower multicellular organisms. Recently, genome sequencing has shown that some bacteria contain genes coding for truncated NOS proteins; this is consistent with reports of NOS-like activities in bacterial extracts. Biological functions for bacterial NOSs are unknown, but have been presumed to be analogous to their role in mammals. Here we describe a gene in the plant pathogen Streptomyces turgidiscabies that encodes a NOS homologue, and we reveal its role in nitrating a dipeptide phytotoxin required for plant pathogenicity. High similarity between bacterial NOSs indicates a general function in biosynthetic nitration; thus, bacterial NOSs constitute a new class of enzymes. Here we show that the primary function of Streptomyces NOS is radically different from that of mammalian NOS. Surprisingly, mammalian NO signalling and bacterial biosynthetic nitration share an evolutionary origin.

Crystal structure of the N-terminal segment of human eukaryotic translation initiation factor 2alpha.

Eukaryotic translation initiation factor 2alpha (eIF2alpha) is a member of the eIF2 heterotrimeric complex that binds and delivers Met-tRNA(i)(Met) to the 40 S ribosomal subunit in a GTP-dependent manner. Phosphorylation/dephosphorylation of eIF2alpha at Ser-51 is the major regulator of protein synthesis in eukaryotic cells. Here, we report the first structural analysis on eIF2, the three-dimensional structure of a 22-kDa N-terminal portion of human eIF2alpha by x-ray diffraction at 1.9 A resolution. This structure contains two major domains. The N terminus is a beta-barrel with five antiparallel beta-strands in an oligonucleotide binding domain (OB domain) fold. The phosphorylation site (Ser-51) is on the loop connecting beta3 and beta4 in the OB domain. A helical domain follows the OB domain, and the first helix has extensive interactions, including a disulfide bridge, to fix its orientation with respect to the OB domain. The two domains meet along a negatively charged groove with highly conserved residues, indicating a likely site for protein-protein interaction.