Entrainment of circadian rhythms in higher organisms relies on light-sensing proteins that communicate to cellular oscillators composed of delayed transcriptional feedback loops. The principal photoreceptor of the fly circadian clock, Drosophila cryptochrome (dCRY), contains a C-terminal tail (CTT) helix that binds beside a FAD cofactor and is essential for light signaling. Light reduces the dCRY FAD to an anionic semiquinone (ASQ) radical and increases CTT proteolytic susceptibility but does not lead to CTT chemical modification. Additional changes in proteolytic sensitivity and small-angle X-ray scattering define a conformational response of the protein to light that centers at the CTT but also involves regions remote from the flavin center. Reduction of the flavin is kinetically coupled to CTT rearrangement. Chemical reduction to either the ASQ or the fully reduced hydroquinone state produces the same conformational response as does light. The oscillator protein Timeless (TIM) contains a sequence similar to the CTT; the corresponding peptide binds dCRY in light and protects the flavin from oxidation. However, TIM mutants therein still undergo dCRY-mediated degradation. Thus, photoreduction to the ASQ releases the dCRY CTT and promotes binding to at least one region of TIM. Flavin reduction by either light or cellular reductants may be a general mechanism of CRY activation.redox | photolyase | protein-protein interaction

The nucleosome core particle (NCP) is the basic structural unit for genome packaging in eukaryotic cells and consists of DNA wound around a core of eight histone proteins. DNA access is modulated through dynamic processes of NCP disassembly. Partly disassembled structures, such as the hexasome (containing six histones) and the tetrasome (four histones), are important for transcription regulation in vivo. However, the pathways for their formation have been difficult to characterize. We combine time-resolved (TR) small-angle X-ray scattering and TR-FRET to correlate changes in the DNA conformations with composition of the histone core during saltinduced disassembly of canonical NCPs. We find that H2A-H2B histone dimers are released sequentially, with the first dimer being released after the DNA has formed an asymmetrically unwrapped, teardrop-shape DNA structure. This finding suggests that the octasome-to-hexasome transition is guided by the asymmetric unwrapping of the DNA. The link between DNA structure and histone composition suggests a potential mechanism for the action of proteins that alter nucleosome configurations such as histone chaperones and chromatin remodeling complexes.G enome access is highly regulated through the hierarchical organization of proteins and nucleic acids within the cell nucleus. The nucleosome core particle (NCP) is the first level of this hierarchy (1) and contains two dimers of H2A-H2B histones and an (H3-H4) 2 tetramer that is assembled as a dimer of dimers. Around this symmetric octamer core, ∼146 base pairs of dsDNA are wrapped in ∼1.7 superhelical turns (1, 2). The NCP structure physically impedes access to DNA, but is dynamically modulated by numerous mechanisms: posttranslational modification (PTM) of histones, incorporation of histone variants, DNA sequence-dependent effects, and the actions of extrinsic protein factors (e.g., histone chaperones, ATP-dependent remodeling complexes, and histone PTM binding proteins) (3, 4).Studies of the intrinsic properties and dynamics of NCPs are critical for understanding how nuclear machinery gains DNA access in vivo (3,5,6). Insight into the nature of partially unfolded NCP structures has been gleaned from in vitro studies of NCP assembly and disassembly. Intermediate species with partially unwrapped DNA (5, 7), disrupted histone-histone interfaces (8, 9), and dissociation of one (hexasomes) or two (tetrasomes) H2A-H2B dimers have been reported (10-12). Some of these NCP intermediates have been directly connected to chromatin function in vivo. For example, the hexasome is formed by the action of RNA Pol II (13) and the essential histone chaperone FACT (14).In addition to equilibrium studies, the kinetics of nucleosome assembly and disassembly have been characterized by bulk and single-molecule methods, including Förster resonance energy transfer (FRET) (7,8,(15)(16)(17), atomic force microscopy (AFM) (9, 18), force spectroscopy (19-21), and small-angle X-ray scattering (SAXS) (10,22). Many studies focused primarily on specific DNA-histon...

The modulation of DNA accessibility by nucleosomes is a fundamental mechanism of gene regulation in eukaryotes. The nucleosome core particle (NCP) consists of 147 bp of DNA wrapped around a symmetric octamer of histone proteins. The dynamics of DNA packaging and unpackaging from the NCP affect all DNA-based chemistries, but depend on many factors, including DNA positioning sequence, histone variants and modifications. Although the structure of the intact NCP has been studied by crystallography at atomic resolution, little is known about the structures of the partially unwrapped, transient intermediates relevant to nucleosome dynamics in processes such as transcription, DNA replication and repair. We apply a new experimental approach combining contrast variation with time-resolved small angle X-ray scattering (TR-SAXS) to determine transient structures of protein and DNA constituents of NCPs during salt-induced disassembly. We measure the structures of unwrapping DNA and monitor protein dissociation from Xenopus laevis histones reconstituted with two model NCP positioning constructs: the Widom 601 sequence and the sea urchin 5S ribosomal gene. Both constructs reveal asymmetric release of DNA from disrupted histone cores, but display different patterns of protein dissociation. These kinetic intermediates may be biologically important substrates for gene regulation.

DNA is tightly wrapped around histone proteins in nucleosome core particles (NCPs) yet must become accessible for processing in the cell. This accessibility, a key component of transcription regulation, is influenced by the properties of both the histone proteins and the DNA itself. Small angle x-ray scattering with contrast variation is used to examine how sequence variations affect DNA unwrapping from NCPs at different salt concentrations. Salt destabilizes NCPs, populating multiple unwrapped states as many possible unwrapping pathways are explored by the complexes. We apply coarse-grained Monte Carlo methods to generate realistic sequence-dependent unwrapped structures for the nucleosomal DNA with thermal variations. An ensemble optimization method is employed to determine the composition of the overall ensemble as electrostatic interactions are weakened. Interesting DNA-sequence-dependent differences are revealed in the unwrapping paths and equilibrium constants. These differences are correlated with specific features within the nucleic acid sequences.

Human pyruvate kinase isoform M2 (PKM2) is a glycolytic enzyme isoform implicated in cancer. Malignant cancer cells have higher levels of dimeric PKM2, which is regarded as an inactive form of tetrameric pyruvate kinase. This perceived inactivity has fueled controversy over how the dimeric form of pyruvate kinase might contribute to cancer. Here we investigate enzymatic properties of PKM2G415R, a variant derived from a cancer patient, which we show by size-exclusion chromatography and SAXS to be a dimer that cannot form tetramer in solution. Although PKM2G415R binds to FBP, unlike wildtype this PKM2 variant shows no activation by FBP. In contrast, PKM2G415R is activated by the SAICAR, an endogenous metabolite that we previously showed correlates with increased cell proliferation and promotes protein kinase activity of PKM2. Our results demonstrate an important and unexpected enzymatic activity of the PKM2 dimer that likely has a key role in cancer progression.