Signal transduction mechanisms of cryptochrome

Photolyase and cryptochrome flavoproteins help living organisms manage the deleterious and beneficial effects of sunlight. Photolyase maintains genome integrity by reversing UV-induced DNA damage with near-UV/blue-light, and cryptochromes act as bluelight photosensory receptors to regulate growth in plants and entrainment of circadian rhythms in both plants and animals. Although photolyase and cryptochrome are highly structurally homologous and the photocycle of photolyase is known in great detail, we do not currently understand how cryptochromes signal in response to light. It is hypothesized that cryptochrome, like photolyase, employs light-driven electron transfer to initiate signaling, although the photocycle and other downstream signaling events remain to be described in detail. The studies described here were designed to take advantage of differences and similarities in the known functions of photolyases and cryptochromes in order to characterize the signaling mechanisms of cryptochromes. An examination of the structural and biochemical properties of plant and animal cryptochromes demonstrates that although they evolved independently from functionally distinct photolyase progenitors, they possess several unexpected similarities, demonstrating convergence in the evolution of cryptochromes. The implications of these results for the cryptochrome photocycle are discussed. Metazoan cryptochromes additionally have a critical, light-independent function in the molecular clock that engenders circadian rhythms. Other studies have shown that iv cryptochromes act as transcriptional repressors in the major transcription/translation feedback loop of the clock. I studied the interaction of mammalian cryptochromes with protein phosphatase 5 (PP5) and show that inhibition of PP5 by cryptochrome modulates the activity of the major clock kinase, casein kinase I epsilon. PP5 is required for proper cycling of the clock; therefore, these data provide the first demonstration of the role of a phosphatase in the mammalian circadian clock. Furthermore, they suggest that cryptochromes regulate the molecular clock by both transcriptional and posttranslational mechanisms

Cryptochrome proteins regulate the circadian intracellular behavior and localization of PER2 in mouse suprachiasmatic nucleus neurons.

The ∼20,000 cells of the suprachiasmatic nucleus (SCN), the master circadian clock of the mammalian brain, coordinate subordinate cellular clocks across the organism, driving adaptive daily rhythms of physiology and behavior. The canonical model for SCN timekeeping pivots around transcriptional/translational feedback loops (TTFL) whereby PERIOD (PER) and CRYPTOCHROME (CRY) clock proteins associate and translocate to the nucleus to inhibit their own expression. The fundamental individual and interactive behaviors of PER and CRY in the SCN cellular environment and the mechanisms that regulate them are poorly understood. We therefore used confocal imaging to explore the behavior of endogenous PER2 in the SCN of PER2::Venus reporter mice, transduced with viral vectors expressing various forms of CRY1 and CRY2. In contrast to nuclear localization in wild-type SCN, in the absence of CRY proteins, PER2 was predominantly cytoplasmic and more mobile, as measured by fluorescence recovery after photobleaching. Virally expressed CRY1 or CRY2 relocalized PER2 to the nucleus, initiated SCN circadian rhythms, and determined their period. We used translational switching to control CRY1 cellular abundance and found that low levels of CRY1 resulted in minimal relocalization of PER2, but yet, remarkably, were sufficient to initiate and maintain circadian rhythmicity. Importantly, the C-terminal tail was necessary for CRY1 to localize PER2 to the nucleus and to initiate SCN rhythms. In CRY1-null SCN, CRY1Δtail opposed PER2 nuclear localization and correspondingly shortened SCN period. Through manipulation of CRY proteins, we have obtained insights into the spatiotemporal behaviors of PER and CRY sitting at the heart of the TTFL molecular mechanism

Purification and Characterization of a Type III Photolyase from Caulobacter crescentus †

Photolyase/cryptochrome family is a large family of flavoproteins that encompasses DNA repair proteins, photolyases; and cryptochromes that regulate blue-light dependent growth and development in plants, and light-dependent and light-independent circadian clock-setting in animals. Phylogenetic analysis has revealed a new branch of the family which co-segregates with plant cryptochromes. Here we describe the isolation and characterization of a member of this family named Type III photolyase, from Caulobacter crescentus. Spectroscopic analysis shows that the enzyme contains both the methenyl-tetrahydrofolate photoantenna and the FAD catalytic cofactor. Biochemical analysis shows that it is a bona fide photolyase that repairs cyclobutane pyrimidine dimers. Mutation of an active site Trp to Arg disrupts FAD binding with no measurable effect on MTHF binding. Using enzyme preparations that contain either or both chromophores we were able to determine the efficiency and rate of energy transfer from MTHF to FAD. Photolyase/cryptochrome family is a large family of flavoproteins that encompasses DNA repair proteins, photolyases; and cryptochromes that regulate blue-light dependent growth and development in plants, and light-dependent and light-independent circadian clock-setting in animals. Phylogenetic analysis has revealed a new branch of the family which co-segregates with plant cryptochromes. Here we describe the isolation and characterization of a member of this family named Type III photolyase, from Caulobacter crescentus. Spectroscopic analysis shows that the enzyme contains both the methenyl-tetrahydrofolate photoantenna and the FAD catalytic cofactor. Biochemical analysis shows that it is a bona fide photolyase that repairs cyclobutane pyrimidine dimers. Mutation of an active site Trp to Arg disrupts FAD binding with no measurable effect on MTHF binding. Using enzyme preparations that contain either or both chromophores we were able to determine the efficiency and rate of energy transfer from MTHF to FAD

Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms

Biofilm formation is critical for the infection cycle of Vibrio cholerae. Vibrio exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for V. cholerae biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Orchestration of Circadian Timing by Macromolecular Protein Assemblies

Genetically encoded biological clocks are found broadly throughout eukaryotes and in cyanobacteria, where they generate circadian (about a day) rhythms that allow organisms to anticipate regular environmental changes and align their physiology and behavior with Earth's daily light/dark cycle. In recent years, many have sought to expand our biochemical and structural understanding of the clock proteins that constitute the molecular "cogs" of these biological clocks. These new studies are beginning to reveal how macromolecular assemblies of dedicated clock proteins form and evolve to contribute to the generation of clocks that function over the timescale of a day. This review will highlight structural and biochemical studies that provide important insight into the molecular mechanisms of cyanobacterial and vertebrate animal clocks. Collectively, these studies demonstrate emerging biochemical properties that appear to be shared by these different clocks, suggesting that there may be some conservation in the regulation and assembly of circadian macromolecular assemblies

Analysis of Protein Stability and Ligand Interactions by Thermal Shift Assay

Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye

Emerging models for the molecular basis of mammalian circadian timing.

Mammalian circadian timekeeping arises from a transcription-based feedback loop driven by a set of dedicated clock proteins. At its core, the heterodimeric transcription factor CLOCK:BMAL1 activates expression of Period, Cryptochrome, and Rev-Erb genes, which feed back to repress transcription and create oscillations in gene expression that confer circadian timing cues to cellular processes. The formation of different clock protein complexes throughout this transcriptional cycle helps to establish the intrinsic ∼24 h periodicity of the clock; however, current models of circadian timekeeping lack the explanatory power to fully describe this process. Recent studies confirm the presence of at least three distinct regulatory complexes: a transcriptionally active state comprising the CLOCK:BMAL1 heterodimer with its coactivator CBP/p300, an early repressive state containing PER:CRY complexes, and a late repressive state marked by a poised but inactive, DNA-bound CLOCK:BMAL1:CRY1 complex. In this review, we analyze high-resolution structures of core circadian transcriptional regulators and integrate biochemical data to suggest how remodeling of clock protein complexes may be achieved throughout the 24 h cycle. Defining these detailed mechanisms will provide a foundation for understanding the molecular basis of circadian timing and help to establish new platforms for the discovery of therapeutics to manipulate the clock