Carrie L. Partch

Molecular Architecture of the Mammalian Circadian Clock

Circadian clocks coordinate physiology and behavior with the 24h solar day to provide temporal homeostasis with the external environment. The molecular clocks that drive these intrinsic rhythmic changes are based on interlocked transcription/translation feedback loops that integrate with diverse environmental and metabolic stimuli to generate internal 24h timing. In this review we highlight recent advances in our understanding of the core molecular clock and how it utilizes diverse transcriptional and post-transcriptional mechanisms to impart temporal control onto mammalian physiology. Understanding the way in which biological rhythms are generated throughout the body may provide avenues for temporally directed therapeutics to improve health and prevent disease.

Crystal Structure of the Heterodimeric CLOCK:BMAL1 Transcriptional Activator Complex

A Timely Structure The physiology and behavior of most organisms are inextricably aligned with the day/night cycle. In mammals, these daily rhythms are generated by a circadian clock encoded by transcriptional activators and repressors operating in a feedback loop that takes about 24 hours to complete. A key participant in this loop is a heterodimeric transcriptional activator consisting of the CLOCK and BMAL1 proteins. Huang et al. (p. 189, published online 31 May; see Perspective by Crane) determined the crystal structure of a complex containing the PAS domains (implicated in protein-protein interactions) and the basic helix-loop-helix domains (implicated in DNA binding) from each protein. CLOCK and BMAL1 were observed to be tightly intertwined in an unusual asymmetric conformation, which may contribute to the stability and activity of the complex. Structure-function analyses reveal details of the interaction between two proteins that regulate daily rhythms in mammals. The circadian clock in mammals is driven by an autoregulatory transcriptional feedback mechanism that takes approximately 24 hours to complete. A key component of this mechanism is a heterodimeric transcriptional activator consisting of two basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domain protein subunits, CLOCK and BMAL1. Here, we report the crystal structure of a complex containing the mouse CLOCK:BMAL1 bHLH-PAS domains at 2.3 Å resolution. The structure reveals an unusual asymmetric heterodimer with the three domains in each of the two subunits—bHLH, PAS-A, and PAS-B—tightly intertwined and involved in dimerization interactions, resulting in three distinct protein interfaces. Mutations that perturb the observed heterodimer interfaces affect the stability and activity of the CLOCK:BMAL1 complex as well as the periodicity of the circadian oscillator. The structure of the CLOCK:BMAL1 complex is a starting point for understanding at an atomic level the mechanism driving the mammalian circadian clock.

Antibacterial membrane attack by a pore-forming intestinal C-type lectin.

Human body-surface epithelia coexist in close association with complex bacterial communities and are protected by a variety of antibacterial proteins. C-type lectins of the RegIII family are bactericidal proteins that limit direct contact between bacteria and the intestinal epithelium and thus promote tolerance to the intestinal microbiota. RegIII lectins recognize their bacterial targets by binding peptidoglycan carbohydrate, but the mechanism by which they kill bacteria is unknown. Here we elucidate the mechanistic basis for RegIII bactericidal activity. We show that human RegIIIα (also known as HIP/PAP) binds membrane phospholipids and kills bacteria by forming a hexameric membrane-permeabilizing oligomeric pore. We derive a three-dimensional model of the RegIIIα pore by docking the RegIIIα crystal structure into a cryo-electron microscopic map of the pore complex, and show that the model accords with experimentally determined properties of the pore. Lipopolysaccharide inhibits RegIIIα pore-forming activity, explaining why RegIIIα is bactericidal for Gram-positive but not Gram-negative bacteria. Our findings identify C-type lectins as mediators of membrane attack in the mucosal immune system, and provide detailed insight into an antibacterial mechanism that promotes mutualism with the resident microbiota.

Structural basis of the day-night transition in a bacterial circadian clock

Molecular clockwork from cyanobacteria The cyanobacterial circadian clock oscillator can be reconstituted in a test tube from just three proteins—KaiA, KaiB, and KaiC—and adenosine triphosphate (ATP). Tseng et al. studied crystal and nuclear magnetic resonance structures of complexes of the oscillator proteins and their signaling output proteins and tested the in vivo effects of structure-based mutants. Large conformational changes in KaiB and ATP hydrolysis by KaiC are coordinated with binding to output protein, which couples signaling and the day-night transitions of the clock. Snijder et al. provide complementary analysis of the oscillator proteins by mass spectrometry and cryo–electron microscopy. Their results help to explain the structural basis for the dynamic assembly of the oscillator complexes. Science, this issue p. 1174, p. 1181 Cyanobacteria make a clock from just three proteins. Circadian clocks are ubiquitous timing systems that induce rhythms of biological activities in synchrony with night and day. In cyanobacteria, timing is generated by a posttranslational clock consisting of KaiA, KaiB, and KaiC proteins and a set of output signaling proteins, SasA and CikA, which transduce this rhythm to control gene expression. Here, we describe crystal and nuclear magnetic resonance structures of KaiB-KaiC,KaiA-KaiB-KaiC, and CikA-KaiB complexes. They reveal how the metamorphic properties of KaiB, a protein that adopts two distinct folds, and the post–adenosine triphosphate hydrolysis state of KaiC create a hub around which nighttime signaling events revolve, including inactivation of KaiA and reciprocal regulation of the mutually antagonistic signaling proteins, SasA and CikA.

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.

Regulating the ARNT/TACC3 axis: Multiple approaches to manipulating protein/protein interactions with small molecules

For several well-documented reasons, it has been challenging to develop artificial small molecule inhibitors of protein/protein complexes. Such reagents are of particular interest for transcription factor complexes given links between their misregulation and disease. Here we report parallel approaches to identify regulators of a hypoxia signaling transcription factor complex, involving the ARNT subunit of the HIF (Hypoxia Inducible Factor) activator and the TACC3 (Transforming Acidic Coiled Coil Containing Protein 3) coactivator. In one route, we used in vitro NMR and biochemical screening to identify small molecules that selectively bind within the ARNT PAS (Per-ARNT-Sim) domain that recruits TACC3, identifying KG-548 as an ARNT/TACC3 disruptor. A parallel, cell-based screening approach previously implicated the small molecule KHS101 as an inhibitor of TACC3 signaling. Here, we show that KHS101 works indirectly on HIF complex formation by destabilizing both TACC3 and the HIF component HIF-1α. Overall, our data identify small molecule regulators for this important complex and highlight the utility of pursuing parallel strategies to develop protein/protein inhibitors.

Cancer/Testis Antigen PASD1 Silences the Circadian Clock

The circadian clock orchestrates global changes in transcriptional regulation on a daily basis via the bHLH-PAS transcription factor CLOCK:BMAL1. Pathways driven by other bHLH-PAS transcription factors have a homologous repressor that modulates activity on a tissue-specific basis, but none have been identified for CLOCK:BMAL1. We show here that the cancer/testis antigen PASD1 fulfills this role to suppress circadian rhythms. PASD1 is evolutionarily related to CLOCK and interacts with the CLOCK:BMAL1 complex to repress transcriptional activation. Expression of PASD1 is restricted to germline tissues in healthy individuals but can be induced in cells of somatic origin upon oncogenic transformation. Reducing PASD1 in human cancer cells significantly increases the amplitude of transcriptional oscillations to generate more robust circadian rhythms. Our results describe a function for a germline-specific protein in regulation of the circadian clock and provide a molecular link from oncogenic transformation to suppression of circadian rhythms.

Coiled-coil Coactivators Play a Structural Role Mediating Interactions in Hypoxia Inducible Factor Heterodimerization

Background: Coiled-coil coactivators can enhance HIF-dependent gene transcription via direct interaction with the HIF/ARNT heterodimer. Results: ARNT uses the β-sheet of the PAS-B domain to recruit coiled-coil coactivators. Conclusion: Coiled-coil coactivators bridge HIF and ARNT via the PAS-B domain β-sheet contacts to both proteins to form a ternary structure. Significance: This work reveals the mechanism for assembling a coiled-coil coactivator complex with the HIF-2 transcription factor heterodimer. The hypoxia-inducible factor complex (HIF-α·aryl hydrocarbon receptor nuclear translocator (ARNT)) requires association with several transcription coactivators for a successful cellular response to hypoxic stress. In addition to the conventional global transcription coactivator CREB-binding protein/p300 (CBP/p300) that binds to the HIF-α transactivation domain, a new group of transcription coactivators called the coiled-coil coactivators (CCCs) interact directly with the second PER-ARNT-SIM (PAS) domain of ARNT (ARNT PAS-B). These less studied transcription coactivators play essential roles in the HIF-dependent hypoxia response, and CCC misregulation is associated with several forms of cancer. To better understand CCC protein recruitment by the heterodimeric HIF transcription factor, we used x-ray crystallography, NMR spectroscopy, and biochemical methods to investigate the structure of the ARNT PAS-B domain in complex with the C-terminal fragment of a coiled-coil coactivator protein, transforming acidic coiled-coil coactivator 3 (TACC3). We found that the HIF-2α PAS-B domain also directly interacts with TACC3, motivating an NMR data-derived model suggesting a means by which TACC3 could form a ternary complex with HIF-2α PAS-B and ARNT PAS-B via β-sheet/coiled-coil interactions. These findings suggest that TACC3 could be recruited as a bridge to cooperatively mediate between the HIF-2α PAS-B·ARNT PAS-B complex, thereby participating more directly in HIF-dependent gene transcription than previously anticipated.

Animal Cryptochromes: Divergent Roles in Light Perception, Circadian Timekeeping and Beyond

Cryptochromes are evolutionarily related to the light‐dependent DNA repair enzyme photolyase, serving as major regulators of circadian rhythms in insects and vertebrate animals. There are two types of cryptochromes in the animal kingdom: Drosophila‐like CRYs that act as nonvisual photopigments linking circadian rhythms to the environmental light/dark cycle, and vertebrate‐like CRYs that do not appear to sense light directly, but control the generation of circadian rhythms by acting as transcriptional repressors. Some animals have both types of CRYs, while others possess only one. Cryptochromes have two domains, the photolyase homology region (PHR) and an extended, intrinsically disordered C‐terminus. While all animal CRYs share a high degree of sequence and structural homology in their PHR domains, the C‐termini are divergent in both length and sequence identity. Recently, cryptochrome function has been shown to extend beyond its pivotal role in circadian clocks, participating in regulation of the DNA damage response, cancer progression and glucocorticoid signaling, as well as being implicated as possible magnetoreceptors. In this review, we provide a historical perspective on the discovery of animal cryptochromes, examine similarities and differences of the two types of animal cryptochromes and explore some of the divergent roles for this class of proteins.

A Slow Conformational Switch in the BMAL1 Transactivation Domain Modulates Circadian Rhythms

The C-terminal transactivation domain (TAD) of BMAL1 (brain and muscle ARNT-like 1) is a regulatory hub for transcriptional coactivators and repressors that compete for binding and, consequently, contributes to period determination of the mammalian circadian clock. Here, we report the discovery of two distinct conformational states that slowly exchange within the dynamic TAD to control timing. This binary switch results from cis/trans isomerization about a highly conserved Trp-Pro imide bond in a region of the TAD that is required for normal circadian timekeeping. Both cis and trans isomers interact with transcriptional regulators, suggesting that isomerization could serve a role in assembling regulatory complexes inxa0vivo. Toward this end, we show that locking the switch into the trans isomer leads to shortened circadian periods. Furthermore, isomerization is regulated by the cyclophilin family of peptidyl-prolyl isomerases, highlighting the potential for regulation of BMAL1 protein dynamics in period determination.