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Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase, RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive due to uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65-substrate complex clarified we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.

Toxin-antitoxin (TA) systems are broadly distributed, yet poorly conserved, genetic elements whose biological functions are unclear and controversial. Some TA systems may provide bacteria with immunity to infection by their ubiquitous viral predators, the bacteriophage. To identify such TA systems, we searched bioinformatically for those frequently encoded near known phage defense genes in bacterial genomes. This search identified homologs of DarTG, a recently discovered family of TA systems whose biological functions and natural activating conditions were unclear. Representatives from two different subfamilies, DarTG1 and DarTG2, strongly protected E. coli MG1655 against different phages. We demonstrate that for each system, infection with either RB69 or T5 phage, respectively, triggers release of the DarT toxin, a DNA ADPribosyltransferase, that then modifies viral DNA and prevents replication, thereby blocking the production of mature virions. Further, we isolated phages that have evolved to overcome DarTG defense either through mutations to their DNA polymerase or to an anti-DarT factor, gp61.2, encoded by many T-even phages. Collectively, our results indicate that phage defense may be a common function for TA systems and reveal the mechanism by which DarTG systems inhibit phage infection.

Carotenoid cleavage oxygenases (CCOs) are non-heme iron enzymes that catalyze scission of alkene groups in carotenoids and stilbenoids to form biologically important products. CCOs possess a rare four-His iron center whose resting-state structure and interaction with substrates are incompletely understood. Here, we address this knowledge gap through a comprehensive structural and spectroscopic study of three phyletically diverse CCOs. The crystal structure of a fungal stilbenoid-cleaving CCO, CAO1, reveals strong similarity between its iron center and those of carotenoid-cleaving CCOs, but with a markedly different substrate-binding cleft. These enzymes all possess a five-coordinate high-spin Fe(II) center with resting-state Fe-His bond lengths of ~2.15 Å. This ligand set generates an iron environment more electropositive than those of other non-heme iron dioxygenases as observed by Mössbauer isomer shifts. Dioxygen (O2) does not coordinate iron in the absence of substrate. Substrates bind away (~4.7 Å) from the iron and have little impact on its electronic structure, thus excluding coordination-triggered O2 binding. However, substrate binding does perturb the spectral properties of CCO Fe–NO derivatives, indicating proximate organic substrate and O2-binding sites, which might influence Fe–O2 interactions. Together, these data provide a robust description of the CCO iron center and its interactions with substrates and substrate mimetics that illuminates commonalities as well as subtle and profound structural differences within the CCO family.

Macrodomain (MD), a highly conserved protein fold present in a subset of plus-strand RNA viruses, binds to and hydrolyzes ADP-ribose (ADPr) from ADPribosylated proteins. ADPr-binding by the alphavirus nonstructural protein 3 (nsP3) MD is necessary for the initiation of virus replication in neural cells, whereas hydrolase activity facilitates replication complex amplification. To determine the importance of these activities for pathogenesis of alphavirus encephalomyelitis, mutations were introduced into the nsP3 MD of Sindbis virus (SINV), and the effects on ADPr binding and hydrolase activities, virus replication, immune responses, and disease were assessed. Elimination of ADPr-binding and hydrolase activities (G32E) severely impaired in vitro replication of SINV in neural cells and in vivo replication in the central nervous systems of 2-week-old mice with reversion to wild type (WT) (G) or selection of a less compromising change (S) during replication. SINVs with decreased binding and hydrolase activities (G32S and G32A) or with hydrolase deficiency combined with better ADPr-binding (Y114A) were less virulent than WT virus. Compared to the WT, the G32S virus replicated less well in both the brain and spinal cord, induced similar innate responses, and caused less severe disease with full recovery of survivors, whereas the Y114A virus replicated well, induced higher expression of interferon-stimulated and NF-B-induced genes, and was cleared more slowly from the spinal cord with persistent paralysis in survivors. Therefore, MD function was important for neural cell replication both in vitro and in vivo and determined the outcome from alphavirus encephalomyelitis in mice. IMPORTANCE Viral encephalomyelitis is an important cause of long-term disability, as well as acute fatal disease. Identifying viral determinants of outcome helps in assessing disease severity and developing new treatments. Mosquito-borne alphaviruses infect neurons and cause fatal disease in mice. The highly conserved macrodomain of nonstructural protein 3 binds and can remove ADP-ribose (ADPr) from ADPribosylated proteins. To determine the importance of these functions for virulence, recombinant mutant viruses were produced. If macrodomain mutations eliminated ADPr-binding or hydrolase activity, viruses did not grow. If the binding and hydrolase activities were impaired, the viruses grew less well than the wild-type virus, induced similar innate responses, and caused less severe disease, and most of the infected mice recovered. If binding was improved, but hydrolase activity was decreased, the virus replicated well and induced greater innate responses than did the WT, but clearance from the nervous system was impaired, and mice remained paralyzed. Therefore, macrodomain function determined the outcome of alphavirus encephalomyelitis. Citation Abraham R, McPherson RL, Dasovich M, Badiee M, Leung AKL, Griffin DE. 2020. Both ADP-ribosyl-binding and hydrolase activities of the alphavirus nsP3 macrodomain affect neurovirulence in mice. mBio 11:e03253-1...

RPE65 is a retinoid isomerase essential for rod function, but its contribution to cone vision is enigmatic. Using selective RPE65 inhibitors, Kiser et al. demonstrate that cone function depends only partially on continuous RPE65 activity, providing support for cone-specific regeneration mechanisms.

Modulators of the visual cycle have been developed for treatment of various retinal disorders. These agents were designed to inhibit retinoid isomerase [retinal pigment epithelium-specific 65 kDa protein (RPE65)], the rate-limiting enzyme of the visual cycle, based on the idea that attenuation of visual pigment regeneration could reduce formation of toxic retinal conjugates. Of these agents, certain ones that contain primary amine groups can also reversibly form retinaldehyde Schiff base adducts, which contributes to their retinal protective activity. Direct inhibition of RPE65 as a therapeutic strategy is complicated by adverse effects resulting from slowed chromophore regeneration, whereas effective retinal sequestration can require high drug doses with potential off-target effects. We hypothesized that the RPE65-emixustat crystal structure could help guide the design of retinaldehydesequestering agents with varying degrees of RPE65 inhibitory activity. We found that addition of an isopropyl group to the central phenyl ring of emixustat and related compounds resulted in agents effectively lacking in vitro retinoid isomerase inhibitory activity, whereas substitution of the terminal 6-membered ring with branched moieties capable of stronger RPE65 interaction potentiated inhibition. The isopropyl derivative series produced discernible visual cycle suppression in vivo, albeit much less potently than compounds with a high affinity for the RPE65 active site. These agents were distributed into the retina and formed Schiff base adducts with retinaldehyde. Except for one compound [3-amino-1-(3-isopropyl-5-((2,6,6-trimethylcyclohex-1-en-1-yl)methoxy)phenyl)propan-1-ol (MB-007)], these agents conferred protection against retinal phototoxicity, suggesting that both direct RPE65 inhibition and retinal sequestration are mechanisms of potential therapeutic relevance.