The Arg-293 of Cryptochrome1 is responsible for the allosteric regulation of CLOCK-CRY1 binding in circadian rhythm

Seref Gul,Cihan Aydin,Onur Ozcan,Berke Gurkan,Saliha Surme,Ibrahim Baris,Ibrahim Halil Kavakli

doi:10.1074/jbc.ra120.014333

Abstract

Mammalian circadian clocks are driven by transcription/translation feedback loops composed of positive transcriptional activators (BMAL1 and CLOCK) and negative repressors (CRYPTOCHROMEs (CRYs) and PERIODs (PERs)). CRYs, in complex with PERs, bind to the BMAL1/CLOCK complex and repress E-box-driven transcription of clock-associated genes. There are two individual CRYs, with CRY1 exhibiting higher affinity to the BMAL1/CLOCK complex than CRY2. It is known that this differential binding is regulated by a dynamic serine-rich loop adjacent to the secondary pocket of both CRYs, but the underlying features controlling loop dynamics are not known. Here we report that allosteric regulation of the serine-rich loop is mediated by Arg-293 of CRY1, identified as a rare CRY1 SNP in the Ensembl and 1000 Genomes databases. The p.Arg293His CRY1 variant caused a shortened circadian period in a Cry1-/-Cry2-/- double knockout mouse embryonic fibroblast cell line. Moreover, the variant displayed reduced repressor activity on BMAL1/CLOCK driven transcription, which is explained by reduced affinity to BMAL1/CLOCK in the absence of PER2 compared with CRY1. Molecular dynamics simulations revealed that the p.Arg293His CRY1 variant altered a communication pathway between Arg-293 and the serine loop by reducing its dynamicity. Collectively, this study provides direct evidence that allosterism in CRY1 is critical for the regulation of circadian rhythm.

Highlights

The circadian clock modulates numerous behavioral and physiological processes through rhythmic transcriptional regulation [1, 2]
The initial transcriptional activation step generates the positive arm of the circadian clock, the latter inhibitory step generates the negative arm of the feedback loop [9, 16]
Mutational analysis of CRYs indicated that residues around the secondary pocket of CRY1 and CRY2 are critical for the CLOCK binding and their repression activity [31]

Summary

Introduction

The circadian clock modulates numerous behavioral and physiological processes through rhythmic transcriptional regulation [1, 2]. Transcriptional factors BMAL1 and CLOCK, having basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains, are central to circadian biology. They form heterodimer and bind to E-box elements (CACGTG), where they initiate the transcription of clock-controlled genes including the Period (Per) and Cryptochrome (Cry) [9,10,11]. CRY1 interacts with cystathionine b-synthase by regulating one-carbon and trans-sulfuration pathways [24] All these studies show functional differences between the CRYs in the circadian clock mechanism. Allosteric regulation in Cryptochrome analyses show that the HI loop on the PAS-B domain of CLOCK docks into the secondary pocket of CRYs [28]. A human gain-of-function CRY1 variant (exon 11 skipping mutation in C-tail of CRY1) found in people suffering from familial delayed sleep phase disorder and attention deficit/hyperactivity disorder exhibits high affinity to BMAL1/ CLOCK [32, 33]

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