User:Linnea Saunders/Sandbox 1

Overview

The circadian locomotor output cycles protein kaput and brain and muscle ARNT-like 1 protein (CLOCK:BMAL1) transcriptional activator complex is an important protein involved in the regulation of the circadian rhythm. The protein shown at the right is a crystallized structure of this complex from Mus musculus. This protein is a heterodimer of CLOCK and BMAL1. Its main function is to interact with DNA at regulatory elements to upregulate the production of proteins period (PER) and cryptochrome (CRY) during the day. These proteins (PER and CRY) heterodimerize at night and interact with CLOCK:BMAL1 to repress the transcription of PER and CRY.¹ They are then degraded, allowing their transcription to occur again. This process takes approximately 24 hours and is the main mechanism of the circadian rhythm, and CLOCK:BMAL1 lies at the heart of this process.² In addition to regulating PER and CRY, CLOCK:BMAL1 regulates the transcription of Rev-erbα, which is a nuclear hormone receptor.³ Rev-erbα once transcribed, inhibits the transcription of BMAL1. CLOCK:BMAL1 contains two necessary domain structures to facilitate its function in the cell, as well as sites for phosphorylation, sumoylation, and ubiquitination, which allow for dimerization, increasing transcriptional activity, and degradation of the complex.

Disease

CLOCK:BMAL1 is known to be a factor in hormone-related breast cancer by contributing to cell growth. Estrogen receptor-α (ERα), when stimulated by estrogen, interacts with CLOCK to increase transcriptional activity at genes regulated by CLOCK:BMAL1. This is achieved by promoting sumoylation of CLOCK at .⁴ When the transcriptional activity of these genes is increased, cell growth is stimulated, and more cells are promoted to the S phase of the cell cycle. CLOCK:BMAL1 is still able to interact with aberrant ERα, allowing this stimulation of growth to occur in tumor cells.

Structural highlights

The main domains of interest within the CLOCK:BMAL1 complex are the Per-Arnt-Sim , which are necessary for dimerization, and the , which allows the complex to interact with DNA. Each subunit contains two PAS domains, which interact with the PAS domains of the other subunit to maintain the structure of the complex.

PAS domains are present in a large variety of proteins, and their function is often to sense different stimuli, such as oxygen tension, redox potential, and light intensity, but they can also serve to facilitate protein-protein interaction, as is the case of CLOCkBMAL1.⁵ The amino acid sequence of this domain varies greatly among the proteins it is present in, as well as among different species, but the structure retains similarities. All PAS domains retain a structure similar to . By clicking through the of CLOCK:BMAL1, , , and , you can observe the general structural trends of the PAS domain. While there is variation within amino acids of the PAS domains in the CLOCK:BMAL1 complex among species, the general structure and presence of the PAS domain is highly conserved.

The bHLH domains of each subunit are crucial to the complex's ability to bind DNA and regulate its transcription. When , the strand of DNA is nestled within the two alpha-helices of bHLH domains of the complex, interacting via hydrogen bonds and Van der Waals forces to support the interactions. The alpha-helices interact with the major groove of the DNA. The intention of this interaction is to regulate the transcription of DNA, CLOCK:BMAL1 interacts with the E-box of DNA, promoting transcription of the gene. This structure within the BMAL1 subunit is highly conserved among species, this is necessary for correct interaction with and regulation of DNA transcription.

Post Translational Modifications

CLOCK:BMAL1 contains a number of amino acids that serve as sites for post translational modification (PTM). The modification of these sites have different purposes to alter the function of the complex. As mentioned above in the disease section, the K67 residue is for the sumoylation of the CLOCK subunit to increase transcriptional activity of the genes that CLOCK:BMAL1 regulates. There are a number of within the complex, that alter protein function and stability. Phosphorylation of the dimer increases the transcriptional activity, alters its subcellular location, and decreases the stability of the heterodimer by promoting degradation of the complex. The complex is hypophosphorylated throughout the day and then hyperphosphorylated at night to promote degradation of the complex.⁶ CLOCK:BMAL1 is ubiquitinated in addition to phosphorylated to promote degradation of the complex.

Links to Shown Structures

Mus musculus CLOCK:BMAL1- [1]

Human CLOCK:BMAL1 bHLH domains interacting with DNA- [2]

Photoactive Yellow Protein- [3]