Conformational Change of Tetratricopeptide Repeats Region Triggers Activation of Phytochrome Associated Protein Phosphatase 5

Phytochrome activity is not only controlled by light but also by post-translational modifications, e. g. phosphorylation. One of the phosphatases responsible for plant phytochrome dephosphorylation and thereby increased activity is the phytochrome-associated protein phosphatase 5 (PAPP5). We show that PAPP5 recognizes phospho-site mimicking mutants of phytochrome B, when being activated by arachidonic acid (AA). Addition of AA to PAPP5 decreases the α-helical content as tracked by CD-spectroscopy. These changes correspond to conformational changes of the regulatory tetratricopeptide repeats (TPR) region as shown by mapping data from hydrogen deuterium exchange mass spectrometry onto a 3.0 Å crystal structure of PAPP5. Surprisingly, parts of the linker between the TPR and PP2A domains and of the so-called C-terminal inhibitory motif exhibit reduced deuterium uptake upon AA-binding. Molecular dynamics analyses of PAPP5 complexed to a phyB phosphopeptide show that this C-terminal motif remains associated with the TPR region in the substrate bound state, suggesting that this motif merely serves for restricting the orientations of the TPR region relative to the catalytic PP2A domain. Given the high similarity to mammalian PP5 these data from a plant ortholog show that the activation mode of these PPP-type protein phosphatases is highly conserved

Nicotinamide Adenine Dinucleotides Arrest Photoreduction of Class II DNA

All light sensitive members of the photolyase cryptochrome family rely on FAD as catalytic cofactor. Its activity is regulated by photoreduction, a light triggered electron transfer process from a conserved tryptophan triad to the flavin. The stability of the reduced flavin depends on available external electron donors and oxygen. In this study, we show for the class II photolyase of Methanosarcina mazei, MmCPDII, that it utilizes physiologically relevant redox cofactors NADH and NADPH for the formation of the semiquinoid FAD in a light dependent reaction. Using redox inert variants MmCPDII W388F and MmCPDII W360F, we demonstrate that photoreduction by NADH and NADPH requires the class II specific tryptophan cascade of MmCPDII. Finally, we confirmed that mutations in the tryptophan cascade can be introduced without any substantial structural disturbances by analyzing crystal structures of MmCPDII W388F, MmCPDII W360F and MmCPDII Y345

Structure of a Native Like Aureochrome 1A Lov Domain Dimer from Phaeodactylum Tricornutum

Light oxygen voltage LOV domains absorb blue light for mediating various biological responses in all three domains of life. Aureochromes from stramenopile algae represent a subfamily of photoreceptors that differs by its inversed topology with a C terminal LOV sensor and an N terminal effector basic region leucine zipper, bZIP domain. We crystallized the LOV domain including its flanking helices, A amp; 8242; amp; 945; and J amp; 945;, of aureochrome 1a from Phaeodactylum tricornutum in the dark state and solved the structure at 2.8 resolution. Both flanking helices contribute to the interface of the native like dimer. Small angle X ray scattering shows light induced conformational changes limited to the dimeric envelope as well as increased flexibility in the lit state for the flanking helices. These rearrangements are considered to be crucial for the formation of the light activated dimer. Finally, the LOV domain of the class 2 aureochrome PtAUREO2 was shown to lack a chromophore because of steric hindrance caused by M30

Functional reprogramming of Candida glabrataepithelial adhesins the role of conserved and variable structural motifs in ligand binding

For host cell interaction, the human fungal pathogen Candida glabrata harbors a large family of more than 20 cell wall attached epithelial adhesins Epas . Epa family members are lectins with binding pockets containing several conserved and variable structural hot spots, which were implicated in mediating functional diversity. In this study, we have performed an elaborate structure based mutational analysis of numerous Epa paralogs to generally determine the role of diverse structural hot spots in conferring host cell binding and ligand binding specificity. Our study reveals that several conserved structural motifs contribute to efficient host cell binding. Moreover, our directed motif exchange experiments reveal that the variable loop CBL2 is key for programming ligand binding specificity, albeit with limited predictability. In contrast, we find that the variable loop L1 affects host cell binding without significantly influencing the specificity of ligand binding. Our data strongly suggest that variation of numerous structural hot spots in the ligand binding pocket of Epa proteins is a main driver of their functional diversification and evolutio

Structure of the bifunctional cryptochrome aCRY from Chlamydomonas reinhardtii

Photolyases and cryptochromes form an almost ubiquitous family of blue light photoreceptors involved in the repair and maintenance of DNA integrity or regulatory control. We found that one cryptochrome from the green alga Chlamydomonas reinhardtii CraCRY is capable of both, control of transcript levels and the sexual cycle of the alga in a positive germination and negative manner mating ability , as well as catalyzing the repair of UV DNA lesions. Its 1.6 crystal structure shows besides the FAD chromophore an aromatic tetrad that is indispensable in animal like type I cryptochromes for light driven change of their signaling active redox state and formation of a stable radical pair. Given CraCRY s catalytic activity as 6 4 photolyase in vivo and in vitro, we present the first co crystal structure of a cryptochrome with duplex DNA comprising a 6 4 pyrimidine pyrimidone lesion. This 2.9 structure reveals a distinct conformation for the catalytic histidine His1, H357, that challenges previous models of a single photon driven 6 4 photolyase mechanis

Structural basis for promiscuity and specificity during Candida glabrata invasion of host epithelia

The human pathogenic yeast Candida glabrata harbors more than 20 surface-exposed, epithelial adhesins (Epas) for host cell adhesion. The Epa family recognizes host glycans and discriminates between target tissues by their adhesin (A) domains, but a detailed structural basis for ligand-binding specificity of Epa proteins has been lacking so far. In this study, we provide high-resolution crystal structures of the Epa1A domain in complex with different carbohydrate ligands that reveal how host cell mucin-type O-glycans are recognized and allow a structure-guided classification of the Epa family into specific subtypes. Further detailed structural and functional characterization of subtype-switched Epa1 variants shows that specificity is governed by two inner loops, CBL1 and CBL2, involved in calcium binding as well as by three outer loops, L1, L2, and L3. In summary, our study provides the structural basis for promiscuity and specificity of Epa adhesins, which might further contribute to developing anti-adhesive antimycotics and combating Candida colonization