Period Circadian Protein
Created by Diane Rodden
Introduction
The Period Circadian Protein (PER) (PDB ID= 3RTY) plays a major role in negative feedback loops and in regulating gene expression. The protein’s major function is to act as a repressor of circadian transcription. Transcription is the first step of gene expression, where a segment of DNA is made into RNA. Specifically, circadian transcription involves gene expression of circadian rhythm, a biological mechanism that oscillates with a period of 24 hours. Circadian rhythm synchronizes many behavioral and biochemical processes with changes in environmental factors. Many different genes control circadian rhythm. The expression of these genes through transcription is what regulates circadian rhythm. PER’s role is to inhibit this circadian transcription by preventing the per gene from being transcribed. PER does this by working with the protein Timeless (TIM). TIM is essential for the entrainment (synchronization of the internal biological clock rhythm to external time cues) to light-dark cycles. Light degrades the TIM protein, which allows organisms to entrain at dawn to environmental cycles. Light-induced degradation of TIM also promotes elimination of PER. Therefore, TIM must dimerize with PER to prevent PER from being degraded. This allows PER accumulation.
When PER levels are high, PER and TIM dissociate and enter the nucleus. (1) Once in the nucleus, PER binds to and inhibits the CLOCK and CYCLE transcription factors in order to shut off per gene transcription. (2) PER’s structure (PDB ID: 3RTY) was initially explained in Drosophila melanogaster. It belongs to the circadian clock protein family. These proteins control the timing of biological processes such as metabolism, sleep, and cell growth through negative feedback loops.
Molecular Pathway
PER and TIM proteins form dimers to prevent degradation of PER. After the PER-TIM complex reaches a high concentration in the early evening, the complex dissociates and PER and TIM are transported into the nucleus. The translocation of PER into the nucleus is triggered by a rapid increase in dimerization between PER and TIM. In addition, the interplay of a nuclear localization signal (NLS) and a cytoplasmic localization domain (CLD) help regulate nuclear entry of PER. (3) In the nucleus PER binds to CLOCK and CYCLE transcription factors. The per gene is activated when these transcription factors are bound to the gene’s promoters. (4) However, PER temporarily inactivates the gene and therefore inhibits transcription by removing the CLK and CYC transcription factors from the promoters. (Fig. 1) The result is a negative feedback loop: the product of the per gene, the PER protein, inhibits its own synthesis. When the concentration of PER reaches low levels, inhibition is lifted and PER activity begins again. This negative feedback loop and the oscillating levels of PER create a circadian rhythm of approximately 24 hours.
Conservation and Homologues
PER is a highly conserved protein. The conservation of the protein sequence is indicated by the presence of identical amino acid residues. Sequence similarities serve as evidence for evolutionary relationships as well as for functional importance. The most highly conserved sequences usually represent major protein domains such as active sites of enzymes and binding sites of protein receptors. In PER, the PAS domain is highly conserved. The PAS domain was named after the three proteins in which it exits: PER, Arnt (Ah receptor nuclear translocator protein), and Sim (single-minded protein). The PAS domain plays an important role as a sensory module for oxygen tension, redox potential, and light. PAS proteins sense the environment by detecting changes in the electron transport system. The PAS domain functions through protein-protein interactions in response to these environmental stimuli. More specifically, t
he PAS domain serves an important role for PER function: PER dimerizes with TIM through the PAS domain repeat region. (1)
PER1, PER2, and PER3 are all mammalian homologues of the Drosophila melanogaster PER. These homologues are similar to PER because they form dimers and negatively regulate circadian expression. In addition, they share sequence similarity with PER in the PAS domain region. However, there also exists two major differences between PER and its homologues. First, these proteins dimerize with cryptochrome proteins (CRY1 and CRY2) instead of with TIM to inhibit transcription. (5) Second, unlike Drosophila PER, NLS is absent in the amino acid sequences of PER1 and PER2. The absence of NLS sequences in these two homologues suggests that they are dependent on interactions with another protein, PER3. PER3 is the homologue most similar to PER because both of these proteins have a functional CLD and NLS. (6)
Structure
The structure of PER is made up of 8 chains with a total number of 2680 residues. The two associated PAS domains are important repeating regions in the protein which allow for the dimerization of PER with TIM. This central repeating structure is a 346-residue fragment made up of two PAS domains (PAS-A and PAS-B). Lys-281 and Asp-418 are two important residues that create intra-molecular interactions between the two PAS domains by forming a salt bridge. Each domain has five antiparallel β-sheet strands surrounded by four α-helices. Two of these alpha helices are on one side of the β-sheet (αA- αA* and αB) and two are on the other (αC and αD). Two other important helices, αE and αF, link the two PAS-AB domains together in order to form a PAS-AB dimer. (Fig.2) Therefore, the entire crystal structure of PER shows four dimers, each containing two PAS-AB repeats. (1)
Mutations
Mutations in the PER protein can dramatically affect clock function by shortening, lengthening, or abolishing circadian rhythms. (Fig.3) One classic circadian mutant, PER long (perL), lengthens the circadian cycle from 24 hours to 28 hours and even abolishes rhythms at high temperatures. This mutation is caused by one residue substitution in the PAS region of PER (V243D). (1) Another classic point mutation, PER short (pers), is caused by the mutation S589N, and shortens the circadian cycle from 24 hours to 19 hours. (7) A third point mutation in PER, which occurs at M560D, prohibits PER homodimerization. In addition, M560 causes a delay in the nuclear entry of PER and diminishes repressor activity. (1)
Summary
PER’s function is to inhibit circadian transcription. Its important PAS domain allows PER to dimerize with TIM, a protein involved in circadian entrainment to light-dark cycles. At high PER levels, PER enters the nucleus with the help of TIM, binds to CLOCK and CYCLE transcription factors, and inactivates the per gene. If the per gene is not transcribed, it is not expressed, and therefore no PER protein is produced. This negative feedback loop means that PER inhibits its own synthesis. Inhibition is lifted when PER concentration levels become low. The rise and fall of PER concentration levels creates this circadian rhythm. The circadian clock is therefore regulated by gene expression and protein regulation. Mutations in the PER protein can also alter circadian clock function by shortening, lengthening, or even abolishing circadian rhythms. In conclusion, PER’s key role as a circadian clock protein is to control the timing of metabolism, sleep and cell growth through negative feedback loops.