59 research outputs found
Structural Characterization Of Human Uch37
Uch37 is a de-ubiquitylating enzyme that is functionally linked with the 26S proteasome via Rpn13, and is essential for metazoan development. Here, we report the X-ray crystal structure of full-length human Uch37 at 2.95 Ã… resolution. Uch37\u27s catalytic domain is similar to those of all UCH enzymes characterized to date. The C-terminal extension is elongated, predominantly helical and contains coiled coil interactions. Additionally, we provide an initial characterization of Uch37\u27s oligomeric state and identify a systematic error in previous analyses of Uch37 activity. Taken together, these data provide a strong foundation for further analysis of Uch37\u27s several functions
Photosensing and Thermosensing by Phytochrome B Require Both Proximal and Distal Allosteric Features within the Dimeric Photoreceptor
Phytochromes (Phys) encompass a diverse collection of bilin-containing photoreceptors that help plants and microorganisms perceive light through photointerconversion between red light (Pr) and far-red light (Pfr)-absorbing states. In addition, Pfr reverts thermally back to Pr via a highly enthalpic process that enables temperature sensation in plants and possibly other organisms. Through domain analysis of the Arabidopsis PhyB isoform assembled recombinantly, coupled with measurements of solution size, photoconversion, and thermal reversion, we identified both proximal and distal features that influence all three metrics. Included are the downstream C-terminal histidine kinase-related domain known to promote dimerization and a conserved patch just upstream of an N-terminal Period/Arnt/Sim (PAS) domain, which upon removal dramatically accelerates thermal reversion. We also discovered that the nature of the bilin strongly influences Pfr stability. Whereas incorporation of the native bilin phytochromobilin into PhyB confers robust Pfr → Pr thermal reversion, that assembled with the cyanobacterial version phycocyanobilin, often used for optogenetics, has a dramatically stabilized Pfr state. Taken together, we conclude that Pfr acquisition and stability are impacted by a collection of opposing allosteric features that inhibit or promote photoconversion and reversion of Pfr back to Pr, thus allowing Phys to dynamically measure light, temperature, and possibly time
Phytochrome B integrates light and temperature signals in Arabidopsis
Ambient temperature regulates many aspects of plant growth and development, but its sensors are unknown. Here, we demonstrate that the phytochrome B (phyB) photoreceptor participates in temperature perception through its temperature-dependent reversion from the active Pfr state to the inactive Pr state. Increased rates of thermal reversion upon exposing Arabidopsis seedlings to warm environments reduce both the abundance of the biologically active Pfr-Pfr dimer pool of phyB and the size of the associated nuclear bodies, even in daylight. Mathematical analysis of stem growth for seedlings expressing wild-type phyB or thermally stable variants under various combinations of light and temperature revealed that phyB is physiologically responsive to both signals. We therefore propose that in addition to its photoreceptor functions, phyB is a temperature sensor in plants
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Photoreversible interconversion of a phytochrome photosensory module in the crystalline state.
A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in Thermosynechococcus elongatus assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on Fobs - Fobs difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Ã… resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion
Crystal Structure of Deinococcus Phytochrome in the Photoactivated State Reveals a Cascade of Structural Rearrangements during Photoconversion
Phytochromes are photochromic photoreceptors responsible for a myriad of red/far-red light-dependent processes in plants and microorganisms. Interconversion is initially driven by photoreversible isomerization of bilin, but how this alteration directs the photostate-dependent changes within the protein to actuate signaling is poorly understood. Here, we describe the structure of the Deinococcus phytochrome photosensory module in its near complete far-red light-absorbing Pfr state. In addition to confirming the 180° rotation of the D-pyrrole ring, the dimeric structure clearly identifies downstream rearrangements that trigger large-scale conformational differences between the dark-adapted and photoactivated states. Mutational analyses verified the importance of residues surrounding the bilin in Pfr stabilization, and protease sensitivity assays corroborated photostate alterations that propagate along the dimeric interface. Collectively, these data support a cooperative toggle model for phytochrome photoconversion and advance our understanding of the allosteric connection between the photosensory and output modules
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