Biology subjects

Ricci, C. G.

Publications and source records attributed to Ricci, C. G..

3 recordsLinked to original sources

bioRxiv2022

PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period

PERIOD (PER) and Casein Kinase 1{delta} regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1{delta} phosphorylation of the Familial Advanced Sleep Phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. Here, we show that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1{delta}. Co-crystal structures in conjunction with accelerated molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1{delta}. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortens circadian period in human cells. We found that Drosophila PER also regulates CK1{delta} via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.

biochemistry↗

bioRxiv2019

Casein kinase 1 dynamics underlie the PER2 circadian phosphoswitch

Post-translational control of PERIOD stability by Casein Kinase 1{delta} and {varepsilon} (CK1) plays a key regulatory role in metazoan circadian rhythms. Despite the deep evolutionary conservation of CK1 in eukaryotes, little is known about its regulation and the factors that influence substrate selectivity on functionally antagonistic sites in PERIOD that directly control circadian period. Here we describe a molecular switch involving a highly conserved anion binding site in CK1. This switch controls conformation of the activation loop to define substrate selectivity on mammalian PER2, thereby directly regulating its stability. Integrated experimental and computational studies shed light on the allosteric linkage between two anion binding sites that dynamically regulate kinase activity. We show that period-altering kinase mutations from humans to Drosophila differentially modulate this activation loop switch to elicit predictable changes in PER2 stability, providing a foundation to understand and further manipulate CK1 regulation of circadian rhythms.

biochemistry↗

bioRxiv2018

Molecular mechanism of off-target effects in CRISPR-Cas9

CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations - using Gaussian accelerated MD (GaMD) - are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at specific distal sites with respect to the Protospacer Adjacent Motif (PAM) induce an extended opening of the RNA:DNA heteroduplex, which leads to newly discovered interactions between the unwound nucleic acids and the protein counterpart. The conserved interactions between the target DNA strand and the L2 loop of the catalytic HNH domain constitute a \"lock\" effectively decreasing the conformational freedom of the HNH domain and its activation for cleavage. Remarkably, depending on their position at PAM distal sites, DNA mismatches leading to off-target cleavages are unable to \"lock\" the HNH domain, thereby identifying the ability to \"lock\" HNH as a key determinant. Consistently, off-target sequences hampering the catalysis have been shown to \"trap\" somehow the HNH domain in an inactive \"conformational checkpoint\" state (Dagdas et al. Sci Adv, 2017). As such, this mechanism identifies the molecular basis underlying off-target cleavages and contributes in clarifying a long-lasting open issue of the CRISPR-Cas9 function. It also poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the \"locking\" interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

biophysics↗