No abstract

The basic helix–loop–helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ∼24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here we show that CRY1 binds directly to the PAS domain core of CLOCK:BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solution X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1.

Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.

Nuclear hormone receptors (NRs) regulate physiology by sensing lipophilic ligands and adapting cellular transcription appropriately. A growing understanding of the impact of circadian clocks on mammalian transcription has sparked interest in the interregulation of transcriptional programs. Mammalian clocks are based on a transcriptional feedback loop featuring the transcriptional activators circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1), and transcriptional repressors cryptochrome (CRY) and period (PER). CRY1 and CRY2 bind independently of other core clock factors to many genomic sites, which are enriched for NR recognition motifs. Here we report that CRY1/2 serve as corepressors for many NRs, indicating a new facet of circadian control of NR-mediated regulation of metabolism and physiology, and specifically contribute to diurnal modulation of drug metabolism.

The AAA+ ATPase spastin remodels microtubule arrays through severing, and its mutation is the most common cause of hereditary spastic paraplegias (HSP). Polyglutamylation of the tubulin C-terminal tail recruits spastin to microtubules and modulates severing activity. Here, we present a ~3.2 Å resolution cryo-EM structure of the Drosophila melanogaster spastin hexamer with a polyglutamate peptide bound in its central pore. Two electropositive loops arranged in a double-helical staircase coordinate the substrate sidechains. The structure reveals how concurrent nucleotide and substrate binding organizes the conserved spastin pore loops into an ordered network that is allosterically coupled to oligomerization, and suggests how tubulin tail engagement activates spastin for microtubule disassembly. This allosteric coupling may apply generally in organizing AAA+ protein translocases into their active conformations. We show that this allosteric network is essential for severing and is a hotspot for HSP mutations.

No abstract

No abstract

The suprachiasmatic nucleus (SCN) defines 24 h of time via a transcriptional/posttranslational feedback loop in which transactivation of Per (period) and Cry (cryptochrome) genes by BMAL1-CLOCK complexes is suppressed by PER-CRY complexes. The molecular/structural basis of how circadian protein complexes function is poorly understood. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutation, early doors (Edo), in the PER-ARNT-SIM (PAS) domain dimerization region of period 2 (PER2) (I324N) that accelerates the circadian clock of Per2 Edo/Edo mice by 1.5 h. Structural and biophysical analyses revealed that Edo alters the packing of the highly conserved interdomain linker of the PER2 PAS core such that, although PER2 Edo complexes with clock proteins, its vulnerability to degradation mediated by casein kinase 1e (CSNK1E) is increased. The functional relevance of this mutation is revealed by the ultrashort (<19 h) but robust circadian rhythms in Per2 Edo/Edo ; Csnk1e Tau/Tau mice and the SCN. These periods are unprecedented in mice. Thus, Per2 Edo reveals a direct causal link between the molecular structure of the PER2 PAS core and the pace of SCN circadian timekeeping.C ircadian rhythms, as exemplified by the sleep/wake cycle, are the outward manifestation of a 24-h timing mechanism that coordinates many physiological processes (1). The principal circadian clock in mammals is the suprachiasmatic nucleus (SCN). At a molecular level, the SCN clockwork consists of interacting positive and negative transcriptional/translational feedback loops (TTFLs) that drive rhythms in the RNA and protein levels of key clock components. Heterodimers of the PER-ARNT-SIM (PAS) domain-containing transcriptional activators CLOCK and BMAL1 drive rhythmic transcription of Per (period) and Cry (cryptochrome) genes by binding to E-box elements. PER proteins associate with CRY, translocating to the nucleus to inhibit their own transcription by interacting with the CLOCK-BMAL1 complex (2, 3). An additional feedback loop involves the nuclear orphan receptors REV-ERBα (4) and RAR-related orphan receptor A (RORA) (5) that modify the transcription of Bmal1, thereby stabilizing and amplifying the CLOCK-BMAL complex-dependent oscillation. Once suppressed, CLOCK-BMAL complex-mediated transactivation can only reoccur after PER and CRY have been degraded.The circadian TTFL is therefore sustained by timely synthesis and degradation of PER and CRY proteins. Changes in PER stability in flies (6, 7) and changes in PER or CRY stability in mammals lead to a faster or slower clock (8). Phosphorylation-dependent licensing of PER proteins for ubiquitination and subsequent proteasomal degradation is a sensitive checkpoint in setting clock speed (9-11). Moreover, two familial advanced sleep phase syndromes (FASPSs) are associated with site-specific phosphorylation of human period 2 (PER2) (12, 13). Finally, the Tau mutation of casein kinase 1e (Csnk1e) (14) is a hypermorph that accelerates the clock of rodents (15). Thus, phosphorylation, PER protein sta...