Biology subjects

Jogl, G.

Publications and source records attributed to Jogl, G..

6 recordsLinked to original sources

bioRxiv2026

Orthosteric and allosteric effects of anti-CRISPR II-C1 inhibition on GeoCas9 from integrated structural biophysics

Anti-CRISPRs (Acrs) are small protein inhibitors of CRISPR-Cas effectors that originate from the translated genetic material of bacteriophage. Harnessing the natural ability of Acrs to bind and disrupt CRISPR-Cas editing can provide enhanced spatiotemporal control of gene editing. Recent studies have revealed diverse structures and functions of Acrs, however, atomistic studies of the specific molecular mechanisms behind Acr inhibition are lacking. Here, we reveal how structure, function, and dynamics govern AcrIIC1 inhibition of Cas9 from G. stearothermophilus (GeoCas9) via its HNH nuclease domain. An X-ray crystal structure of the GeoHNH-AcrIIC1complex reveals a conserved binding interface at the catalytic site and disruption of crucial electrostatic contacts known to modulate the thermostability of GeoCas9. AcrIIC1 binding also rewires the intrinsic dynamics of the GeoHNH domain, stimulates millisecond motions that are absent from the unliganded nuclease, and attenuates the guide RNA affinity of GeoCas9. Subsequent AcrIIC1 mutations in residues at its crystallographic binding interface uncouple Acr binding from inhibition, providing new insight into mechanism by which AcrIIC1 acts on GeoCas9.

biophysics↗

bioRxiv2024

Atomistic Tuning of the GeoCas9 Recognition Lobe Modulates Allosteric Motions and Guide RNA Interactions

The intuitive manipulation of specific amino acids to alter the activity or specificity of CRISPR-Cas9 has been a topic of great interest. As a large multi-domain RNA-guided endonuclease, the intricate molecular crosstalk within the Cas9 protein hinges on its conformational dynamics, but a comprehensive understanding of the extent and timescale of the motions that drive its allosteric function and association with nucleic acids remains elusive. Here, we investigated the structure and multi-timescale molecular motions of the recognition (Rec) lobe of GeoCas9, a thermophilic Cas9 from Geobacillus stearothermophilus. Our results provide new atomic details about the GeoRec subdomains (GeoRec1, GeoRec2) and the full-length domain in solution. Two rationally designed mutants, K267E and R332A, enhanced and redistributed micro-millisecond flexibility throughout GeoRec, and NMR studies of the interaction between GeoRec and its guide RNA showed that mutations reduced this affinity and the stability of the ribonucleoprotein complex. Despite measured biophysical differences due to the mutations, DNA cleavage assays reveal no functional differences in on-target activity, and similar specificity. These data suggest that guide RNA interactions can be tuned at the biophysical level in the absence of major functional losses but also raise questions about the underlying mechanism of GeoCas9, since analogous single-point mutations have significantly impacted on- and off-target DNA editing in mesophilic S. pyogenes Cas9. A K267E/R332A double mutant did also did not enhance GeoCas9 specificity, highlighting the robust tolerance of mutations to the Rec lobe of GeoCas9 and species-dependent complexity of Rec across Cas9 paralogs. Ultimately, this work provides an avenue by which to modulate the structure, motion, and guide RNA interactions at the level of the Rec lobe of GeoCas9, setting the stage for future studies of GeoCas9 variants and their effect on its allosteric mechanism.

biophysics↗

bioRxiv2023

High-Fidelity, Hyper-Accurate, and Evolved Mutants Rewire Atomic Level Communication in CRISPR-Cas9

The Cas9-HF1, HypaCas9, and evoCas9 variants of the Cas9 endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in the Rec3 domain mediate specificity in these variants are poorly understood. Here, solution NMR and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and peculiar dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic HNH domain. The variants remodel the communication sourcing from the Rec3 -helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles.

biophysics↗

bioRxiv2022

Identification and characterization of small molecule inhibitors of the LINE-1 retrotransposon endonuclease

The long interspersed nuclear element-1 (LINE-1 or L1) retrotransposon is the only active autonomously replicating retrotransposon in the human genome. While most of the approximately 500,000 L1 copies are no longer able to propagate, those that retain activity can harm the cell by creating mutations, generating DNA damage, and triggering the expression of inflammatory factors such as the host interferon anti-viral response. Therefore, inhibition of L1 could be used to treat a variety of diseases associated with these processes. Previous research has focused on inhibition of the L1 reverse transcriptase (RT) activity, in part due to the prevalence of well-characterized existing inhibitors to related viral enzymes. Here we present the L1 endonuclease (EN) as an additional target for reducing the detrimental effects of L1 expression. We have screened and characterized a set of structurally diverse small molecule EN inhibitors using computational, biochemical, and cellular methods. We also show that these inhibitors reduce DNA damage created by L1 and inflammation reinforced by L1 activity in senescent cells. These inhibitors could be further used to modulate endogenous L1 function to better understand the lifecycle of this ubiquitous disease-relevant element.

biochemistry↗

bioRxiv2020

Phase separation of the LINE-1 ORF1 protein is mediated by the N-terminus and coiled-coil domain

Long Interspersed Nuclear Element-1 (LINE-1 or L1) is a retrotransposable element that autonomously replicates in the human genome, resulting in DNA damage and genomic instability. Activation of L1 in senescent cells triggers a type I interferon response and age-associated inflammation. Two open reading frames encode an ORF1 protein functioning as mRNA chaperone and an ORF2 protein providing catalytic activities necessary for retrotransposition. No function has been identified for the conserved, disordered N-terminal region of ORF1. Using microscopy and NMR spectroscopy, we demonstrate that ORF1 forms liquid droplets in vitro in a salt-dependent manner and that interactions between its N-terminal region and coiled-coil domain are necessary for phase separation. Mutations disrupting blocks of charged residues within the N-terminus impair phase separation while some mutations within the coiled-coil domain enhance phase separation. Demixing of the L1 particle from the cytosol may provide a mechanism to protect the L1 transcript from degradation. Statement of significanceOver half of the human genome is comprised of repetitive sequences. The Long Interspersed Nuclear Element-1 (L1) is an autonomous mobile DNA element that can alter its genomic location, resulting in genomic instability and DNA damage. L1 encodes two proteins that are required for this function: the ORF1 RNA chaperone and the enzymatic ORF2. Here, we demonstrate that ORF1 forms liquid-liquid phase separated states in vitro, which is mediated by electrostatic interactions between the conserved, disordered N-terminus and coiled-coil domain. This work provides a framework to explore how L1 phase separation may enhance the ability of the retrotransposable element to colonize the genome by preventing degradation of the L1 transcript and evasion of host immune responses.

biophysics↗

bioRxiv2019

Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics

CRISPR-Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and a novel Gaussian accelerated Molecular Dynamics (GaMD) simulation method are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond timescale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in full-length CRISPR-Cas9. These findings reveal a potential route of signal transduction within the CRISPR-Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR-Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome editing capability.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC=\"FIGDIR/small/660613v4_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (64K):\norg.highwire.dtl.DTLVardef@13429c5org.highwire.dtl.DTLVardef@1a2493borg.highwire.dtl.DTLVardef@175e120org.highwire.dtl.DTLVardef@f1852_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗