Biology subjects

Shin, Y.-C.

Publications and source records attributed to Shin, Y.-C..

4 recordsLinked to original sources

bioRxiv2025

Spacer Extension Reconciles Specificity and Activity in High-Fidelity Cas9 Genome Editing

AbstractEngineering high-fidelity CRISPR enzymes often leads to reduced cleavage activity, creating a significant hurdle in balancing nuclease specificity and efficiency for clinical applications. Here, we demonstrate that extending the spacer to 21 or 22 nucleotides restores the impaired cleavage activity of SuperFi-Cas9, an engineered high-fidelity Cas9 variant with seven mutations at the PAM-distal region. Structural and mutational analyses reveal that the spacer extension strengthens additional interactions at the PAM-distal end, stabilizing the nuclease- sgRNA-DNA complex, which appears to be disturbed due to the seven mutations. This approach not only provides a high-fidelity Cas9 with uncompromised efficiency but also introduces a novel strategy to enhance CRISPR complex stability. Our findings offer a promising avenue for precise and efficient genome editing, crucial for advancing CRISPR technologies toward clinical translation.

genomics↗

bioRxiv2024

Structural basis of respiratory complexes adaptation to cold temperatures

In response to cold, mammals activate brown fat for respiratory-dependent thermogenesis reliant on the electron transport chain (1, 2). Yet, the structural basis of respiratory complex adaptation to cold remains elusive. Herein we combined thermoregulatory physiology and cryo-EM to study endogenous respiratory supercomplexes exposed to different temperatures. A cold-induced conformation of CI:III2 (termed type 2) was identified with a [~]25{degrees} rotation of CIII2 around its inter-dimer axis, shortening inter-complex Q exchange space, and exhibiting different catalytic states which favor electron transfer. Large-scale supercomplex simulations in lipid membrane reveal how unique lipid-protein arrangements stabilize type 2 complexes to enhance catalytic activity. Together, our cryo-EM studies, multiscale simulations and biochemical analyses unveil the mechanisms and dynamics of respiratory adaptation at the structural and energetic level.

cell biology↗

bioRxiv2023

The crystal and cryo-EM structures of PLCγ2 reveal dynamic inter-domain recognitions in autoinhibition

Abstract/SummaryPhospholipase C gamma 2 (PLC{gamma}2) plays important roles in cell signaling downstream of various membrane receptors. PLC{gamma}2 contains a multi-domain inhibitory region critical for its regulation, while it has remained unclear how these domains contribute to PLC{gamma}2 activity modulation. Here we determined three structures of human PLC{gamma}2 in autoinhibited states, which reveal dynamic interactions at the autoinhibition interface, involving the conformational flexibility of the SH3 domain in the inhibitory region, and its previously unknown interaction with a C-terminal helical domain in the core region. We also determined a structure of PLC{gamma}2 bound to the kinase domain of fibroblast growth factor receptor 1 (FGFR1), which demonstrates the recognition of FGFR1 by the nSH2 domain in the inhibitory region of PLC{gamma}2. Our results provide new structural insights into PLC{gamma}2 regulation that will facilitate future mechanistic studies to understand the entire activation process.

biochemistry↗

bioRxiv2023

Structural basis for auto-inhibition and activation of a short prokaryotic Argonaute associated TIR-APAZ defense system

Short prokaryotic Ago accounts for most prokaryotic Argonaute (pAgo) and is involved in defending bacteria against invading nucleic acids. Short prokaryotic Ago associated with APAZ-TIR (SPARTA) has been shown to oligomerize and deplete NAD+ upon guide-mediated target DNA recognition. However, the molecular basis of SPARTA inhibition and activation remains unknown. Here we determine the cryo-EM structures of Crenotalea thermophila SPARTA in its inhibited, transient, as well as activated states. The SPARTA is auto-inhibited by its acidic tail, which occupies the guide-target binding channel. Guide mediated target binding expels this acidic tail and triggers substantial conformational changes to expose Ago-Ago dimerization interface. As a result, SPARTA assembles into an active TIR-APAZ4/short Ago4 octamer, where the four TIR domains are rearranged and packed to form NADase active sites. Together with biochemical evidence, our results provide a panoramic vision explaining SPARTA auto-inhibition and activation, and expand understanding of pAgo mediated bacterial defense systems.

biochemistry↗