Biology subjects

Novick, S. J.

Publications and source records attributed to Novick, S. J..

4 recordsLinked to original sources

bioRxiv2022

Molecular glue CELMoD compounds are allosteric regulators of cereblon conformation

Cereblon (CRBN) is an ubiquitin ligase (E3) adaptor protein co-opted by CRBN E3 Ligase Modulatory Drugs (CELMoD) agents that target therapeutically-relevant proteins for degradation. Prior crystallographic studies defined the drug-binding site within CRBNs Thalidomide Binding Domain (TBD), but the allostery of drug-induced neosubstrate binding remains unclear. We therefore performed cryo-EM analyses of the DDB1[~]CRBN apo-complex, and compared these structures with DDB1[~]CRBN in the presence of CELMoD compounds alone and complexed with neosubstrates. Association of CELMoD compounds to the TBD is necessary and sufficient for triggering CRBN allosteric rearrangement from an "open" conformation to the canonical "closed" conformation. Importantly, the neosubstrate Ikaros only stably associates with the closed CRBN conformation, illustrating the importance of allostery for CELMoD compound efficacy, and informing structure-guided design strategies to improve therapeutic efficacy.

molecular biology↗

bioRxiv2021

Conformational Changes of RORγ During Response Element Recognition and Coregulator Engagement.

The retinoic acid receptor-related orphan receptor {gamma} (ROR{gamma}) is a ligand-dependent transcription factor of the nuclear receptor super family that underpins metabolic activity, immune function, and cancer progression. Despite being a valuable drug target in health and disease, our understanding of the ligand-dependent activities of ROR{gamma} is far from complete. Like most nuclear receptors, ROR{gamma} must recruit coregulatory protein to enact the ROR{gamma} target gene program. To date, a majority of structural studies have been focused exclusively on the ROR{gamma} ligand-binding domain and the ligand-dependent recruitment of small peptide segments of coregulators. Herein, we examine the ligand-dependent assembly of full length ROR{gamma}:coregulator complexes on cognate DNA response elements using structural proteomics and small angle x-ray scattering. The results from our studies suggest that ROR{gamma} becomes elongated upon DNA recognition, preventing long range interdomain crosstalk. We also determined that the DNA binding domain adopts a sequence-specific conformation, and that coregulatory proteins may be able to sense the ligand- and DNA-bound status of ROR{gamma}. We propose a model where ligand-dependent coregulator recruitment may be influenced by the sequence of the DNA to which ROR{gamma} is bound. Overall, the efforts described herein will illuminate important aspects of full length ROR{gamma} and monomeric orphan nuclear receptor target gene regulation through DNA-dependent conformational changes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/445650v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1f0a067org.highwire.dtl.DTLVardef@b2e9f6org.highwire.dtl.DTLVardef@1e15d5org.highwire.dtl.DTLVardef@12b8546_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Structures of the Human LONP1 Protease Reveal Regulatory Steps Involved in Protease Activation

The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in the pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the molecular mechanism of substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. However, unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate to increase interactions with the translocating peptide as it transits into the proteolytic chamber for proteolysis. Further, we show that substrate-bound LONP1 includes a second level of regulation at the proteolytic active site, wherein autoinhibition of the active site is only relieved by the presence of a peptide substrate. Ultimately, our results define a structural basis for human LONP1 proteolytic activation and activity, establishing a molecular framework to understand the critical importance of this protease for mitochondrial regulation in health and disease.

molecular biology↗

bioRxiv2018

Structural basis for the RNA-guided ribonuclease activity of CRISPR-Cas13d

CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function, we resolved cryo-electron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 [A] resolution, respectively. Furthermore, a 6.5 [A] reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, explain the compact molecular architecture of Cas13d and provide insights into the structural transitions required for enzyme activation. Our comprehensive analysis of Cas13d in diverse enzymatic states facilitated site-specific truncations for minimal size and delineates a blueprint for improving biomolecular applications of RNA targeting.

biochemistry↗