GAF Domain Research Articles

Phytochromobilin Binding and Specific Amino Acid Residues Near The Chromophore Contribute To Orange Light Perception By The Dualchrome Phytochrome Region.

A novel photoreceptor dualchrome 1 (DUC1), containing a fused structure of cryptochrome and phytochrome, was discovered in the marine green alga Pycnococcus provasolli. The DUC1 phytochrome region (PpDUC1-N) binds to the bilin (linear tetrapyrrole) chromophores, phytochromobilin (PΦB) or phycocyanobilin (PCB), and reversibly photoconverts between the orange-absorbing dark-adapted state and the far-red-absorbing photoproduct state. This contrasts with typical phytochromes, which photoconvert between the red-absorbing dark-adapted and far-red-absorbing photoproduct states. In this study, we examined the molecular mechanism of PpDUC1-N to sense orange light by identifying the chromophore species synthesized by P. provasolli and the amino acid residues within the PpDUC1-N responsible for sensing orange light in the dark-adapted state. We focused on the PcyA homolog of P. provasolli (PpPcyA). Coexpression with the photoreceptors followed by an enzymatic assay revealed that PpPcyA synthesized PCB. Next, we focused on the PpDUC1-N GAF domain responsible for chromophore binding and light sensing. Ten amino acid residues were selected as the mutagenesis target near the chromophore. Replacement of these residues with those conserved in typical phytochromes revealed that three mutations (F290Y/M304S/L353M) resulted in a 23-nm red-shift in the dark-adapted state. Finally, we combined these constructs to obtain the PΦB-binding F290Y/M304S/L353M mutant and a 38-nm red-shift was observed compared with the PCB-binding wild-type PpDUC1. The binding chromophore species and the key residues near the chromophore contribute to blue-shifted orange light sensing in the dark-adapted state of the PpDUC1-N.

Teal-light absorbing cyanobacterial phytochrome superfamily provides insights into the diverse mechanisms of spectral tuning and facilitates the engineering of photoreceptors for optogenetic tools

Cyanobacteriochromes (CBCRs) are distinctive tetrapyrrole (bilin)-binding photoreceptors exclusively found in cyanobacteria. Unlike canonical phytochromes, CBCRs require only a GAF (cGMP-phosphodiesterase/adenylate cyclase/FhlA) domain for autolyase activity to form a bilin adduct via a Cys residue and cis-trans photoisomerization. Apart from the canonical Cys, which attaches covalently to C31 in the A-ring of the bilin, some GAF domains of CBCRs contain a second-Cys in the Asp-Xaa-Cys-Phe (DXCF) motif, responsible for isomerization of phycocyanobilin (PCB) to phycoviolobilin (PVB) and/or for the formation of a reversible 2nd thioether linkage to the C10. Unlike green/teal-absorbing GAF proteins lacking ligation activity, the second-Cys in another teal-absorbing lineage (DXCF blue/teal group) exhibits both isomerization and ligation activity due to the presence of the Tyr instead of His next to the canonical Cys. Herein, we discovered an atypical CBCR GAF protein, Tpl7205g1, belonging to the DXCF blue/teal group, but having His instead of Tyr next to the first-Cys. Consistent with its subfamily, the second-Cys of Tpl7205g1 did not form a thioether linkage at C10 of PCB, showing only isomerization activity. Instead of forming 2nd thioether linkage, this novel GAF protein exhibits a pH-dependent photocycle between protonated 15Z and deprotonated 15E. Site-directed mutagenesis to the GAF scaffolds revealed its combined characteristics, including properties of teal-DXCF CBCRs and red/green-absorbing CBCRs (XRG CBCRs), suggesting itself as the evolutionary bridge between the two CBCR groups. Our study thus sheds light on the expanded spectral tuning characteristics of teal-light absorbing CBCRs and enhances feasibility of engineering these photoreceptors.

Crystal structure of the photosensory module from a PAS-less cyanobacterial phytochrome as Pr shows a mix of dark-adapted and photoactivated features

Phytochromes (Phys) are a diverse collection of photoreceptors that regulate numerous physiological and developmental processes in microorganisms and plants through photointerconversion between red-light absorbing Pr and far-red light-absorbing Pfr states. Light is detected by an N-terminal photosensing module (PSM) sequentially comprised of Period/ARNT/Sim (PAS), cGMP-phosphodiesterase/adenylyl cyclase/FhlA (GAF), and Phy-specific (PHY) domains, with the bilin chromophore covalently-bound within the GAF domain. Phys sense light via the Pr/Pfr ratio measured by light-induced rotation of the bilin D-pyrrole ring that triggers conformational changes within the PSM, which for microbial Phys reaches into an output region. A key step is a β-stranded to α-helical reconfiguration of a hairpin loop extending from the PHY domain to contact the GAF domain. Besides canonical Phys, cyanobacteria express several variants, including a PAS-less subfamily that harbors just the GAF and PHY domains for light detection. Prior 2D-NMR studies of a model PAS-less Phy from Synechococcus_sp._JA-2-3B'a(2-13) (SyB-Cph1) proposed a unique photoconversion mechanism involving an A-pyrrole ring rotation, while magic-angle-spinning NMR probing the chromophore proposed the prototypic D-ring flip. To help solve this conundrum, we determined the crystallographic structure of the GAF-PHY region from SyB-Cph1 as Pr. Surprisingly, this structure differs from canonical Phys by having a Pr ZZZsyn,syn,anti bilin configuration but shifted to the activated position in the binding pocket with consequent folding of the hairpin loop to α-helical, an architecture common for Pfr. Collectively, the PSM of SyB-Cph1 as Pr displayed a mix of dark-adapted and photoactivated features whose co-planar A-C pyrrole rings supports a D-ring flip mechanism.

Photoreversible Aggregation of the Biliprotein Containing the First and Second GAF Domains of a Cyanobacteriochrome All2699 in Nostoc sp. PCC7120.

As plant photoreceptors, phytochromes are capable of detecting red light and far-red light, thereby governing plant growth. All2699 is a photoreceptor found in Nostoc sp. PCC7120 that specifically responds to red light and far-red light. All2699g1g2 is a truncated protein carrying the first and second GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains of All2699. In this study, we found that, upon exposure to red light, the protein underwent aggregation, resulting in the formation of protein aggregates. Conversely, under far-red light irradiation, these protein aggregates dissociated. We delved into the factors that impact the aggregation of All2699g1g2, focusing on the protein structure. Our findings showed that the GAF2 domain contains a low-complexity (LC) loop region, which plays a crucial role in mediating protein aggregation. Specifically, phenylalanine at position 239 within the LC loop region was identified as a key site for the aggregation process. Furthermore, our research revealed that various factors, including irradiation time, temperature, concentration, NaCl concentration, and pH value, can impact the aggregation of All2699g1g2. The aggregation led to variations in Pfr concentration depending on temperature, NaCl concentration, and pH value. In contrast, ΔLC did not aggregate and therefore lacked responses to these factors. Consequently, the LC loop region of All2699g1g2 extended and enhanced sensory properties.

PDE10A Mutation as an Emerging Cause of Childhood-Onset Hyperkinetic Movement Disorders: A Review of All Published Cases.

Cyclic nucleotide phosphodiesterase (PDE) enzymes catalyze the breakdown of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which act as intracellular second messengers for signal transduction pathways and modulate various processes in the central nervous system. Recent discoveries that mutations in genes encoding different PDEs, including PDE10A, are responsible for rare forms of chorea in children led to the recognition of an emerging role of PDEs in the field of pediatric movement disorders. A comprehensive literature review of all reported cases of PDE10A mutations in PubMed and Web of Science was performed in English. We included eight studies, describing 31 patients harboring a PDE10A mutation and exhibiting a hyperkinetic movement disorder with onset in infancy or childhood. Mutations in both GAF-A, GAF-B regulatory domains and outside the GAF domains of the PDE10A gene have been reported to cause hyperkinetic movement disorders. In general, patients with homozygous mutations in either GAF-A domain of PDE10A present with a more severe phenotype and at an earlier age but without any extensive abnormalities of the striata compared with patients with dominant variants in GAF-B domain, indicating that dominant and recessive mutations have different pathogenic mechanisms. PDE10A plays a key role in regulating control of striato-cortical movement. Comprehension of the molecular mechanisms within the cAMP and cGMP signaling systems caused by PDE10A mutations may inform novel therapeutic strategies that could alleviate symptoms in young patients affected by these rare movement disorders.

Crucial Residue for Tuning Thermal Relaxation Kinetics in the Biliverdin-binding Cyanobacteriochrome Photoreceptor Revealed by Site-saturation Mutagenesis

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to the phytochromes sensing red and far-red light reversibly. Only the cGMP phosphodiesterase/Adenylate cyclase/FhlA (GAF) domain is needed for chromophore incorporation and proper photoconversion. The CBCR GAF domains covalently ligate linear tetrapyrrole chromophores and show reversible photoconversion between two light-absorbing states. In most cases, the two light-absorbing states are stable under dark conditions, but in some cases, the photoproduct state undergoes thermal relaxation back to the dark-adapted state during thermal relaxation. In this study, we examined the engineered CBCR GAF domain, AnPixJg2_BV4. AnPixJg2_BV4 covalently binds biliverdin IX-alpha (BV) and shows reversible photoconversion between a far-red-absorbing Pfr dark-adapted state and an orange-absorbing Po photoproduct state. Because the BV is an intrinsic chromophore of mammalian cells and absorbs far-red light penetrating into deep tissues, BV-binding CBCR molecules are useful for the development of optogenetic and bioimaging tools used in mammals. To obtain a better developmental platform molecule, we performed site-saturation random mutagenesis on the Phe319 position. We succeeded in obtaining variant molecules with higher chromophore-binding efficiency and higher molar extinction coefficient. Furthermore, we observed a wide variation in thermal relaxation kinetics, with an 81-fold difference between the slowest and fastest rates. Both molecules with relatively slow and fast thermal relaxation would be advantageous for optogenetic control.

Cyanobacteriochromes from Gloeobacterales Provide New Insight into the Diversification of Cyanobacterial Photoreceptors

The phytochrome superfamily comprises three groups of photoreceptors sharing a conserved GAF (cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and formate hydrogen lyase transcription activator FhlA) domain that uses a covalently attached linear tetrapyrrole (bilin) chromophore to sense light. Knotted red/far-red phytochromes are widespread in both bacteria and eukaryotes, but cyanobacteria also contain knotless red/far-red phytochromes and cyanobacteriochromes (CBCRs). Unlike typical phytochromes, CBCRs require only the GAF domain for bilin binding, chromophore ligation, and full, reversible photoconversion. CBCRs can sense a wide range of wavelengths (ca. 330–750 nm) and can regulate phototaxis, second messenger metabolism, and optimization of the cyanobacterial light-harvesting apparatus. However, the origins of CBCRs are not well understood: we do not know when or why CBCRs evolved, or what selective advantages led to retention of early CBCRs in cyanobacterial genomes. In the current work, we use the increasing availability of genomes and metagenome-assembled-genomes from early-branching cyanobacteria to explore the origins of CBCRs. We reaffirm the earliest branches in CBCR evolution. We also show that early-branching cyanobacteria contain late-branching CBCRs, implicating early appearance of CBCRs during cyanobacterial evolution. Moreover, we show that early-branching CBCRs behave as integrators of light and pH, providing a potential unique function for early CBCRs that led to their retention and subsequent diversification. Our results thus provide new insight into the origins of these diverse cyanobacterial photoreceptors.

Zinc-Induced Fluorescence Turn-On in Native and Mutant Phycoerythrobilin-Binding Orange Fluorescent Proteins.

Cyanobacteriochrome (CBCR)-derived fluorescent proteins are a class of reporters that can bind bilin cofactors and fluoresce across the ultraviolet to the near-infrared spectrum. Derived from phytochrome-related photoreceptor proteins in cyanobacteria, many of these proteins use a single small GAF domain to autocatalytically bind a bilin and fluoresce. The second GAF domain of All1280 (All1280g2) from Nostoc sp. PCC7120 is a DXCF motif-containing protein that exhibits blue-light-responsive photochemistry when bound to its native cofactor, phycocyanobilin. All1280g2 can also bind non-photoswitching phycoerythrobilin (PEB), resulting in a highly fluorescent protein. Given the small size, high quantum yield, and that unlike green fluorescent proteins, bilin-binding proteins can be used in anaerobic organisms, the orange fluorescent All1280g2-PEB protein is a promising platform for designing new genetically encoded metal ion sensors. Here, we show that All1280g2-PEB undergoes a ∼5-fold reversible zinc-induced fluorescence enhancement with a blue-shifted emission maximum (572 to 517 nm), which is not observed for a related PEB-bound GAF from Synechocystis sp. PCC6803 (Slr1393g3). Zn2+ significantly enhances All1280g2-PEB fluorescence across a biologically relevant pH range from 6.0 to 9.0, with pH-dependent dissociation constants from 1 μM to ∼20-80 nM. Site-directed mutants aiming to sterically decrease and increase access to PEB show a decreased and similar amount of zinc-induced fluorescence enhancement. Mutation of the cysteine residue within the DXCF motif to alanine abolishes the zinc-induced fluorescence enhancement. Collectively, these results support the presence of a unique fluorescence-enhancing Zn2+ binding site in All1280g2-PEB likely involving coordination to the bilin cofactor and requiring a nearby cysteine residue.

Time-resolved fluorescence anisotropy with Atto 488-labeled phytochrome Agp1 from Agrobacterium fabrum.

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. Three domains, termed PAS, GAF, and PHY domains, constitute the N-terminal photosensory chromophore module (PCM); the C-terminus is often a histidine kinase module. In the Agrobacterium fabrum phytochrome Agp1, the autophosphorylation activity of the histidine kinase is high in the Pr and low in the Pfr form. Crystal structure analyses of PCMs suggest flexibility around position 308 in the Pr but not in the Pfr form. Here, we performed time-resolved fluorescence anisotropy measurements with different Agp1 mutants, each with a single cysteine residue at various positions. The fluorophore label Atto-488 was attached to each mutant, and time-resolved fluorescence anisotropy was measured in the Pr and Pfr forms. Fluorescence anisotropy curves were fitted with biexponential functions. Differences in the amplitude A2 of the second component between the PCM and the full-length variant indicate a mechanical coupling between position 362 and the histidine kinase. Pr-to-Pfr photoconversion induced no significant changes in the time constant t2 at any position. An intermediate t2 value at position 295, which is located in a compact environment, suggests flexibility around the nearby position 308 in Pr and in Pfr.

Impact of Fe + 2 ions on structural integrity of A0A6P1CI42_RHITR NifA protein from Rhizobium tropici strain CIAT 899

A0A6P1CI42_RHITR, a protein originating from Rhizobium tropici strain CIAT 899, has emerged as a key player in leguminous plant symbiosis and nitrogen fixation processes. Understanding the intricate details of its structure and function holds immense significance for unraveling the molecular mechanisms underlying its biological activities. In this study, we employed molecular modeling and molecular dynamics (MD) simulations to investigate the A0A6P1CI42_RHITR protein, with a specific emphasis on the influence of Fe-atoms, linker structural integrity, and conformational changes within the GAF domain. Our findings unveiled noteworthy conformational changes in the A0A6P1CI42_RHITR protein, particularly within the GAF domain, when Fe-atoms were present compared to its apo form. Significant conformational rearrangements after an initial 20 ns, accompanied by the opening of the ligand substrate accommodating loop in the GAF domain influenced by Fe-atoms was observed. At the residue level, the investigation revealed substantial activity variations in individual residues, particularly in those contributing to the GAF domain from positions 51 to 223. Intriguingly, the presence of Fe-atoms led to controlled movement of conserved cysteine residues at positions 467 and 472, indicating a correlation between interlinker domain motion and the activity of the GAF domain loop responsible for substrate accommodation. Moreover, in the presence of Fe-atoms, the distance between Cys467 and Cys472 residues was maintained, ensuring the overall structural integrity of the interdomain loop necessary for protein activation. Conversely, in the apo form, a sudden flip motion of cysteine residues’ thiol groups was observed, leading to a loss of structural integration. Overall, our study utilizing molecular modeling and MD simulations offers valuable insights into the structural dynamics and functional implications of the A0A6P1CI42_RHITR protein. Communicated by Ramaswamy H. Sarma

Transcriptional regulation of soluble methane monooxygenase via enhancer-binding protein derived from Methylosinus sporium 5.

Methane is a major greenhouse gas, and methanotrophs regulate the methane level in the carbon cycle. Soluble methane monooxygenase (sMMO) is expressed in various methanotroph genera, including Alphaproteobacteria and Gammaproteobacteria, and catalyzes the hydroxylation of methane to methanol. It has been proposed that MmoR regulates the expression of sMMO as an enhancer-binding protein under copper-limited conditions; however, details on this transcriptional regulation remain limited. Herein, we elucidate the transcriptional pathway of sMMO depending on copper ion concentration, which affects the interaction of MmoR and sigma factor. MmoR and sigma-54 (σ54) from Methylosinus sporium 5 were successfully overexpressed in Escherichia coli and purified to investigate sMMO transcription in methanotrophs. The results indicated that σ54 binds to a promoter positioned -24 (GG) and -12 (TGC) upstream between mmoG and mmoX1. The binding affinity and selectivity are lower (Kd = 184.6 ± 6.2 nM) than those of MmoR. MmoR interacts with the upstream activator sequence (UAS) with a strong binding affinity (Kd = 12.5 ± 0.5 nM). Mutational studies demonstrated that MmoR has high selectivity to its binding partner (ACA-xx-TGT). Titration assays have demonstrated that MmoR does not coordinate with copper ions directly; however, its binding affinity to UAS decreases in a low-copper-containing medium. MmoR strongly interacts with adenosine triphosphate (Kd = 62.8 ± 0.5 nM) to generate RNA polymerase complex. This study demonstrated that the binding events of both MmoR and σ54 that regulate transcription in M. sporium 5 depend on the copper ion concentration. IMPORTANCE This study provides biochemical evidence of transcriptional regulation of soluble methane monooxygenase (sMMO) in methanotrophs that control methane levels in ecological systems. Previous studies have proposed transcriptional regulation of MMOs, including sMMO and pMMO, while we provide further evidence to elucidate its mechanism using a purified enhancer-binding protein (MmoR) and transcription factor (σ54). The characterization studies of σ54 and MmoR identified the promoter binding sites and enhancer-binding sequences essential for sMMO expression. Our findings also demonstrate that MmoR functions as a trigger for sMMO expression due to the high specificity and selectivity for enhancer-binding sequences. The UV-visible spectrum of purified MmoR suggested an iron coordination like other GAF domain, and that ATP is essential for the initiation of enhancer elements. Binding assays indicated that these interactions are blocked by the copper ion. These results provide novel insights into gene regulation of methanotrophs.

A Single-Site Mutation Tunes Fluorescence and Chromophorylation of an Orange Fluorescent Cyanobacteriochrome.

Cyanobacteriochrome (CBCR) cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, including the third GAF domain of CBCR Slr1393 from Synechocystis sp. PCC6803, which binds phycoerythrobilin (PEB) to yield a bright orange fluorescent protein. Compared to green fluorescent proteins, the smaller size and lack of an oxygen requirement for fluorescence make Slr1393g3 a promising platform for new genetically encoded fluorescent tools. Slr1393g3, however, shows low PEB binding efficiency (chromophorylation) at ~3 % compared to total Slr1393g3 expressed in E. coli. Here we used site-directed mutagenesis and plasmid redesign methods to improve Slr1393g3-PEB binding and demonstrate its utility as a fluorescent marker in live cells. Mutation at a single site, Trp496, tuned the emission over ~30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications for tuning relative expression of Slr1393g3 and PEB synthesis enzymes also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised up to a total of 23 % with combined sequence truncation and W496H mutation.

Genome-Wide Survey of the RWP-RK Gene Family in Cassava (Manihot esculenta Crantz) and Functional Analysis.

The plant-specific RWP-RK transcription factor family plays a central role in the regulation of nitrogen response and gametophyte development. However, little information is available regarding the evolutionary relationships and characteristics of the RWP-RK family genes in cassava, an important tropical crop. Herein, 13 RWP-RK proteins identified in cassava were unevenly distributed across 9 of the 18 chromosomes (Chr), and these proteins were divided into two clusters based on their phylogenetic distance. The NLP subfamily contained seven cassava proteins including GAF, RWP-RK, and PB1 domains; the RKD subfamily contained six cassava proteins including the RWP-RK domain. Genes of the NLP subfamily had a longer sequence and more introns than the RKD subfamily. A large number of hormone- and stress-related cis-acting elements were found in the analysis of RWP-RK promoters. Real-time quantitative PCR revealed that all MeNLP1-7 and MeRKD1/3/5 genes responded to different abiotic stressors (water deficit, cold temperature, mannitol, polyethylene glycol, NaCl, and H2O2), hormonal treatments (abscisic acid and methyl jasmonate), and nitrogen starvation. MeNLP3/4/5/6/7 and MeRKD3/5, which can quickly and efficiently respond to different stresses, were found to be important candidate genes for further functional assays in cassava. The MeRKD5 and MeNLP6 proteins were localized to the cell nucleus in tobacco leaf. Five and one candidate proteins interacting with MeRKD5 and MeNLP6, respectively, were screened from the cassava nitrogen starvation library, including agamous-like mads-box protein AGL14, metallothionein 2, Zine finger FYVE domain containing protein, glyceraldehyde-3-phosphate dehydrogenase, E3 Ubiquitin-protein ligase HUWE1, and PPR repeat family protein. These results provided a solid basis to understand abiotic stress responses and signal transduction mediated by RWP-RK genes in cassava.

Conformational change in an engineered biliverdin-binding cyanobacteriochrome during the photoconversion process

Cyanobacteriochromes (CBCRs) derived from cyanobacteria are linear-tetrapyrrole-binding photoreceptors related to the canonical red/far-red reversible phytochrome photoreceptors. CBCRs contain chromophore-binding cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that are highly diverse in their primary sequences and are categorized into many subfamilies. Among this repertoire, the biliverdin (BV)-binding CBCR GAF domains receive considerable attention for their in vivo optogenetic and bioimaging applications because BV is a mammalian intrinsic chromophore and can absorb far-red light that penetrates deep into the mammalian body. The typical BV-binding CBCR GAF domain exhibits reversible photoconversion between far-red-absorbing dark-adapted and orange-absorbing photoproduct states. Herein, we applied various biochemical and spectral studies to identify the details of the conformational change during this photoconversion process. No oligomeric state change was observed, whereas the surface charge would change with a modification of the α-helix structures during the photoconversion process. Combinatorial analysis using partial protease digestion and mass spectrometry identified the region where the conformational change occurred. These results provide clues for the future development of optogenetic tools.

Introduction of reversible cysteine ligation ability to the biliverdin-binding cyanobacteriochrome photoreceptor.

Cyanobacteriochrome (CBCR) photoreceptors are distantly related to the canonical red/far-red reversible phytochrome photoreceptors. In the case of the CBCRs, only the GAF domain is required for chromophore incorporation and photoconversion. The GAF domains of CBCR are highly diversified into many lineages to sense various colors of light. These CBCR GAF domains are divided into two types: those possessing only the canonical Cys residue and those with both canonical and second Cys residues. The canonical Cys residue stably ligates to the chromophore in both cases. The second Cys residue mostly shows reversible adduct formation with the chromophore during photoconversion for spectral tuning. In this study, we focused on the CBCR GAF domain AnPixJg2_BV4, which possesses only the canonical Cys residue. AnPixJg2_BV4 covalently ligates to the biliverdin (BV) chromophore and shows far-red/orange reversible photoconversion. Because BV is a mammalian intrinsic chromophore, BV-binding molecules are advantageous for in vivo optogenetic and bioimaging tool development. To obtain a better developmental platform molecule, we performed site-saturation random mutagenesis and serendipitously obtained a unique variant molecule that showed far-red/blue reversible photoconversion, in which the Cys residue was introduced near the chromophore. This introduced Cys residue functioned as the second Cys residue that reversibly ligated with the chromophore. Because the position of the introduced Cys residue is distinct from the known second Cys residues, the variant molecule obtained in this study would expand our knowledge about the spectral tuning mechanism of CBCRs and contribute to tool development.

Development of bright red-shifted miRFP704nano using structural analysis of miRFPnano proteins.

We recently converted the GAF domain of NpR3784 cyanobacteriochrome into near-infrared (NIR) fluorescent proteins (FPs). Unlike cyanobacterichrome, which incorporates phycocyanobilin tetrapyrrole, engineered NIR FPs bind biliverdin abundant in mammalian cells, thus being the smallest scaffold for it. Here, we determined the crystal structure of the brightest blue-shifted protein of the series, miRFP670nano3, at 1.8 Å resolution, characterized its chromophore environment and explained the molecular basis of its spectral properties. Using the determined structure, we have rationally designed a red-shifted NIR FP, termed miRFP704nano, with excitation at 680 nm and emission at 704 nm. miRFP704nano exhibits a small size of 17 kDa, enhanced molecular brightness, photostability and pH-stability. miRFP704nano performs well in various protein fusions in live mammalian cells and should become a versatile genetically-encoded NIR probe for multiplexed imaging across spatial scales in different modalities.

Biochemical characterization of GAF domain of free-R-methionine sulfoxide reductase from Trypanosoma cruzi

Trypanosoma cruzi is the causal agent of Chagas Disease and is a unicellular parasite that infects a wide variety of mammalian hosts. The parasite exhibits auxotrophy by L-Met; consequently, it must be acquired from the extracellular environment of the host, either mammalian or invertebrate. Methionine (Met) oxidation produces a racemic mixture (R and S forms) of methionine sulfoxide (MetSO). Reduction of L-MetSO (free or protein-bound) to L-Met is catalyzed by methionine sulfoxide reductases (MSRs). Bioinformatics analyses identified the coding sequence for a free-R-MSR (fRMSR) enzyme in the genome of T. cruzi Dm28c. Structurally, this enzyme is a modular protein with a putative N-terminal GAF domain linked to a C-terminal TIP41 motif. We performed detailed biochemical and kinetic characterization of the GAF domain of fRMSR in combination with mutant versions of specific cysteine residues, namely, Cys12, Cys98, Cys108, and Cys132. The isolated recombinant GAF domain and full-length fRMSR exhibited specific catalytic activity for the reduction of free L-Met(R)SO (non-protein bound), using tryparedoxins as reducing partners. We demonstrated that this process involves two Cys residues, Cys98 and Cys132. Cys132 is the essential catalytic residue on which a sulfenic acid intermediate is formed. Cys98 is the resolutive Cys, which forms a disulfide bond with Cys132 as a catalytic step. Overall, our results provide new insights into redox metabolism in T. cruzi, contributing to previous knowledge of L-Met metabolism in this parasite.

Expanding the genotype-phenotype landscape of PDE10A-associated movement disorders (S42.004)

<h3>Objective:</h3> To report novel PDE10A genotype-phenotype in four affected family members <h3>Background:</h3> Phosphodiesterase 10A (PDE10A) controls body movements by regulating cyclic adenosine monophosphate signaling in the basal ganglia. Two classes of PDE10A variants are reported with distinctive genotype-phenotype correlation. The autosomal recessive mutations in the GAF-A and catalytic domains are associated with compromised membrane localization, and manifested with infantile onset chorea, developmental, and cognition delay with normal brain MRI. Conversely, autosomal dominant mutations in the GAF-B domain cause protein aggregates which results in childhood onset chorea in the context of normal cognition and development despite striatal nigral lesions. Herein, we report four cases of <i>PDE10A</i>-related chorea in a native Arab family with biallelic mutations in PDE10A. <h3>Design/Methods:</h3> Phenotypic characteristics of affected family members with PDE10A mutations were recorded, in addition to Sanger sequencing and in silico analysis to identify the mutations. <h3>Results:</h3> Four individuals from a consanguineous family affected with PDE10A mutations were observed for up to 40 years. Although these individuals displayed a clinical phenotype attributed to the recessive GAF-A mutations, they revealed an autosomal recessive GAF-B mutation (c.883G&gt;A:p. D295N; p.Asp295Asn) that was segregated from all affected individuals. In addition to chorea, we observed peculiar foot deformities and pronounced social phobia. In silico structural analysis suggested that the GAF-B mutation blocked allosteric PDE10A activation. The resulting lack of PDE10A activity without protein aggregation photocopies GAFA-mutations but is achieved through a distinct mechanism. <h3>Conclusions:</h3> Collectively, our work explains the recessive and dominant phenotypes of known variants and expands the genotype-phenotype landscape of PDE10A-associated movement disorders. <b>Disclosure:</b> Saeed Bohlega has nothing to disclose. Zainah Al- Qahtani has nothing to disclose. Francisco J. Guzmán-Vega has nothing to disclose. Dr. Ramakrishnan has nothing to disclose. Dr. Abusrair has nothing to disclose. Haya Aldosari has nothing to disclose. Amaal AlDakheel has nothing to disclose. Dr. Al Qahtani has nothing to disclose. Dorota Monies has nothing to disclose. Dr. Arold has nothing to disclose.

PtsN in Pseudomonas aeruginosa Is Phosphorylated by Redundant Upstream Proteins and Impacts Virulence-Related Genes.

The bacterial nitrogen-related phosphotransfer (PTSNtr; here, Nitro-PTS) system bears homology to well-known PTS systems that facilitate saccharide import and phosphorylation. The Nitro-PTS comprises an enzyme I (EI), PtsP; an intermediate phosphate carrier, PtsO; and a terminal acceptor, PtsN, which is thought to exert regulatory effects that depend on its phosphostate. For instance, biofilm formation by Pseudomonas aeruginosa can be impacted by the Nitro-PTS, as deletion of either ptsP or ptsO suppresses Pel exopolysaccharide production and additional deletion of ptsN elevates Pel production. However, the phosphorylation state of PtsN in the presence and absence of its upstream phosphotransferases has not been directly assessed, and other targets of PtsN have not been well defined in P. aeruginosa. We show that PtsN phosphorylation via PtsP requires the GAF domain of PtsP and that PtsN is phosphorylated on histidine 68, as in Pseudomonas putida. We also find that FruB, the fructose EI, can substitute for PtsP in PtsN phosphorylation but only in the absence of PtsO, implicating PtsO as a specificity factor. Unphosphorylatable PtsN had a minimal effect on biofilm formation, suggesting that it is necessary but not sufficient for the reduction of Pel in a ptsP deletion. Finally, we use transcriptomics to show that the phosphostate and the presence of PtsN do not appear to alter the transcription of biofilm-related genes but do influence genes involved in type III secretion, potassium transport, and pyoverdine biosynthesis. Thus, the Nitro-PTS influences several P. aeruginosa behaviors, including the production of its signature virulence factors. IMPORTANCE The PtsN protein impacts the physiology of a number of bacterial species, and its control over downstream targets can be altered by its phosphorylation state. Neither its upstream phosphotransferases nor its downstream targets are well understood in Pseudomonas aeruginosa. Here, we examine PtsN phosphorylation and find that the immediate upstream phosphotransferase acts as a gatekeeper, allowing phosphorylation by only one of two potential upstream proteins. We use transcriptomics to discover that PtsN regulates the expression of gene families that are implicated in virulence. One emerging pattern is a repression hierarchy by different forms of PtsN: its phosphorylated state is more repressive than its unphosphorylated state, but the expression of its targets is even higher in its complete absence.

Engineering of bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s.

Optogenetic tools for controlling protein-protein interactions (PPIs) have been developed from a small number of photosensory modules that respond to a limited selection of wavelengths. Cyanobacteriochrome (CBCR) GAF domain variants respond to an unmatched array of colors; however, their natural molecular mechanisms of action cannot easily be exploited for optogenetic control of PPIs. Here we developed bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s by engineering synthetic light-dependent interactors for a red/green GAF domain. The systematic approach enables the future engineering of the broad chromatic palette of CBCRs for optogenetics use. BICYCLs are among the smallest optogenetic tools for controlling PPIs and enable either green-ON/red-OFF (BICYCL-Red) or red-ON/green-OFF (BICYCL-Green) control with up to 800-fold state selectivity. The access to green wavelengths creates new opportunities for multiplexing with existing tools. We demonstrate the utility of BICYCLs for controlling protein subcellular localization and transcriptional processes in mammalian cells and for multiplexing with existing blue-light tools.