Understanding Light-Regulation by CRYPTOCHROME: A Structural Story

Circadian clocks are biochemical oscillators that entrain an organism’s physiology to the period of a day-night cycle. The general architecture of a circadian clock is homologous across species and is simply a transcription-translation feedback loop (TTFL). A TTFL in the context of a circadian clock is composed of positive elements that initiate the transcription of clock-controlled genes, and negative elements that are the clock-controlled gene products (or core oscillator proteins) that inhibit their own expression after some time delay. However, rhythmic binding of photoactive elements and phosphorylation of the core oscillator proteins initiate their respective degradation, thus liberating the inhibition of the positive elements and restarting the cycle. Dysfunction within this cycle has been linked to numerous maladies in humans and animals, including sleep-phase disorders, metabolic diseases, and cancer. The focus of this thesis is the Drosophila melanogaster circadian clock. In brief, the transcription factors Clock and Cycle (CLK and CYC) form a heterodimeric complex to transcribe the clock-controlled genes period (per) and timeless (tim). After a time delay, their respective gene products, PER and TIM, form a heterodimeric complex and translocate into the nucleus to inhibit the CLK/CYC complex from further transcribing per and tim. What entrains the clock to light, however, is the circadian photosensor, Cryptochrome (CRY). In response to blue light, its harbored flavin adenine dinucleotide (FADox) cofactor is photoreduced to the anionic semiquinone state (ASQ), which triggers a conformational change in its C-terminal tail (CTT). This structural event is what then enables CRY to bind TIM in the presence of light, further recruiting an E3 ubiquitin ligase, Jetlag (JET). CRY and TIM are then ubiquitinated and degraded in the proteasomal pathway. CRY is also degraded by another E3 ligase in a light-dependent manner. In this thesis, I present dynamical and structural studies of the Drosophila melanogaster CRY to elucidate details of light-dependent regulation within the clock. Chapter 1 serves as an introduction to the thesis, in which background information on the Drosophila clock, and the structure and function of CRY are the focus. Chapters 2 and 3 focus on utilizing DEER spectroscopy to elucidate the conformational dynamics of the CTT in a proxy-dark and light state. To achieve this, a mutant of CRY was engineered to form the neutral semiquinone (NSQ) acting as a proxy-dark state, and the CTT was selectively spin-labeled using an enzymatically mediated method to ligate a pre-spin-labeled peptide to it. Point mutations at His378, which is thought to mediate CTT undocking in the signal active state, were made in the context of this variant and the WT parent to understand how charge and sterics play a role in CTT undocking. Further, biochemical assays reveal that a neighboring histidine residue, His377, is also strongly involved in TIM binding and CTT undocking in the light. Chapter 4 focuses on solving the structure of CRY at room temperature. The approach taken to doing so was crystallizing CRY in batch and utilizing a novel technique, fixed-target serial synchrotron crystallography, to diffract hundreds of crystals; yielding a dataset diffracting to 3.5 Å. More detailed filtration of the data by filtering out reflections of similar symmetry, overall agreement of mini-datasets against the whole, or how datasets correlate with each other reveal drastic effects on the LLG score of a molecular replacement solution and the overall completeness of the dataset used for refinement; the Rfree post-refinement utilizing one of these strategies also dramatically improves, providing evidence for the argument of quality over quantity in serial data processing. Lastly, chapter 5 will focus on deconvoluting CRY/TIM interactions to further understanding on how CRY recognizes TIM utilizing in vitro techniques. Depending on the altitude at which Drosophila are found, they will possess one of two forms of TIM: 1) short TIM; 2) long TIM (23 residue N-terminal extension). Recently, a cryoEM structure of CRY_ and full-length TIM in complex, where N-terminal residues of TIM were shown to be interacting strongly with various residues within the CRY_ FAD binding pocket. In vivo pulldown assays have shown that CRY_ binds TIM regardless of dark and light, leading to TIM’s degradation in the proteasomal pathway. We sought to understand how the binding affinity, relative to CRY_ and WT for this isoform and how it may differ in the dark and light, which could have use in potential optogenetic applications. Because TIM is a difficult protein to recombinantly express in E. coli due to its size (~120 kDa), an N-terminal peptide of short TIM was cloned in frame with a SUMO-tag in pET28a(+). These peptides were either recombinantly purified or purchased and labeled with fluorescein-5-maleimide, with binding affinity determined by fluorescence anisotropy. The full-length peptide was also compared to the S-TIM with three of the C-terminal residues of the long TIM ortholog and without the initiating methionine residue. Pulldowns utilizing twin-strep-tagged dCRY (1-539) and S-TIM cloned onto the N-terminus of enhanced green fluorescent protein (eGFP) with a spacer were performed in dark and light to verify the ability of dCRY to discriminate against the peptide in light and probe further optogenetic applications.