Biology subjects

Enchev, R. I.

Publications and source records attributed to Enchev, R. I..

7 recordsLinked to original sources

bioRxiv2025

The ribosome synchronizes folding and assembly to promote oligomeric protein biogenesis

Natural proteins are structurally diverse and often form intricate multidomain, oligomeric architectures. This presents a prima facie challenge to cellular homeostasis, as topologically complex proteins seldom refold efficiently in vitro. How cells overcome sequence-intrinsic folding limitations to optimize protein biogenesis is incompletely understood. Here, we show that efficient folding and assembly of the model five-domain homotetramer {beta}-galactosidase is obligatorily coupled to its synthesis on the ribosome, and define the underlying mechanisms. During refolding of full-length protein from denaturant, maturation of the catalytic domain is frustrated. Assembly outpaces monomer folding, and non-native oligomers accumulate. The ribosome directs the order of folding events and specifies the pathway of oligomer assembly. Efficient de novo folding is characterised by segmental domain folding, shaped by binding of a nascent amphipathic helix to a cryptic pocket on the ribosome surface. Homomer assembly initiates cotranslationally via recruitment of a full-length subunit to the nascent polypeptide, and the failure to do so results in misassembly. Our results reveal how the ribosome can dictate the timing of folding and assembly to enable efficient biogenesis of a topologically complex protein.

biochemistry↗

bioRxiv2025

Chronobot: Deep learning guided time-resolved cryo-EM captures molecular choreography of RecA in homology search

The function of proteins and other biological macromolecules is regulated by conformational dynamics1. Many functional changes take place on millisecond timescales which cannot be experimentally captured by manual sample preparation for cryo-EM2. Here we introduce Chronobot, a robust, data-driven platform enabling reproducible, time-resolved cryo-EM sample preparation to visualize these transient intermediates. We quantify ice thickness, an important precondition and thus a reliable predictor of 3D reconstruction quality, using two complementary methods: a bespoke deep learning model analysing high-speed camera videos of the grid just before vitrification, and detailed ice thickness quantifications of outputs from common TEM screening workflows like the EPU software. Combining these methods enables rapid optimisation, resulting in an 11-fold improvement in cryo-EM sample quality compared to our previously reported workflow3. To demonstrate the Chronobot in capturing transient reaction intermediates visualised through cryo-EM and single particle analysis we focused on RecA homology search. The RecA family of recombinases perform the essential task of rapidly scanning for homologous dsDNA sequences to initiate homologous recombination. The dynamics of these RecA-dsDNA interactions occur on millisecond timescales, limiting structural insights4. We capture time-resolved homology search intermediates at 250 milliseconds. These structures reveal the involvement of the secondary DNA binding site in initial capture of dsDNA before homology sampling occurs. We also observe three-strand homology sampling intermediates, where the homologous strand is not fully displaced, and homology is not stably bound. Our results suggest a model of how RecA-family recombinases function in early homologous recombination, by coordinating the incoming DNA between RecAs various DNA binding sites depending on the stage of homology search and the presence of suUicient homology. We anticipate the Chronobot method to be broadly applicable to processes which cannot be captured by manual sample preparation methods. In addition, by leveraging AI inference, our rapid user feedback mechanisms allow for per-sample optimisation of grid conditions, increasing the likelihood of success and reducing the sample requirements of each time-resolved experiment.

biochemistry↗

bioRxiv2025

Structural Basis of CSN-mediated SCF Deneddylation

Cullin-RING ligases (CRLs) are the largest family of E3 ligases, with their ubiquitination activity dynamically regulated by neddylation and deneddylation by the COP9 signalosome (CSN). CSN-mediated deneddylation not only deactivates CRLs but also enables substrate receptor exchange, fine-tuning CRL specificity. CSN has emerged as a promising drug target, yet the structural basis underlying its catalytic mechanism remains insufficiently understood. To address this, we used cryo-electron microscopy (cryo-EM) analysis of CSN-CRL1 (SCF) complexes to uncover distinct functional states, capturing key intermediates of the deneddylation cycle. The earliest state represents an initial docking step in which CSN remains autoinhibited. In contrast, the catalytic intermediate reveals the fully engaged state, with the CSN5 Ins-1 loop, RBX1 RING, and neddylated Cullin WHB domains repositioned for isopeptide bond cleavage. We further resolve four dissociation intermediates that define the stepwise release of CSN from its deneddylated product, highlighting a central role for RBX1RING in stabilising key interactions throughout this process. Additionally, our structures reveal the previously uncharacterised position of CSNAP, which integrates into the CSN scaffold at a groove formed by CSN3 and CSN8. Together, our study provides a comprehensive mechanistic model of CSN function, linking CRL recognition, catalytic activation, and stepwise disengagement. These insights lay the groundwork for the rational design of CSN inhibitors, offering new opportunities to modulate CRL activity for therapeutic applications. Key TakeawaysO_LIHigh-resolution cryo-EM structures capture key intermediates of the CSN-mediated SCF deneddylation cycle C_LIO_LIStructural identification of CSNAP places it within a previously uncharacterized groove at the interface of CSN3 and CSN8. C_LIO_LIPre-catalytic and catalytic states define the conformational transitions of CSN5Ins-1, RBX1RING, and N8WHB that enable isopeptide bond cleavage. C_LIO_LIFour dissociation intermediates reveal a sequential pathway for CSN disengagement, with RBX1RING playing a central role in stabilising key interactions. C_LI

biochemistry↗

bioRxiv2024

GroEL/ES chaperonin unfolds then encapsulates a nascent protein on the ribosome

The bacterial chaperonin GroEL/ES promotes protein folding post-translation by transiently encapsulating substrate proteins within a central chamber. GroEL also binds translating ribosomes in vivo, suggesting an additional role in cotranslational folding. Here, we used biochemical reconstitution, structural proteomics and electron microscopy to study the mechanism by which GroEL/ES engages nascent polypeptides. We show that GroEL binds nascent chains on the inside of its cavity via the apical domains and disordered C-terminal tails, resulting in local structural destabilization of the substrate. Ribosome-tethered nascent proteins are partially encapsulated upon GroES binding to GroEL, and refold in the chaperonin cavity. Reconstitution of chaperone competition at the ribosome shows that both Trigger factor and GroEL can be accommodated on long nascent chains, but GroEL and DnaK are mutually antagonistic. Our findings extend the role of GroEL/ES in de novo protein folding, and reveal an unexpected plasticity of the chaperonin mechanism that allows cotranslational substrate encapsulation.

biochemistry↗

bioRxiv2020

Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins

Homologous recombination (HR) is an essential DNA double-strand break (DSBs) repair mechanism frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated resected DNA and catalyses strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employ single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, while RFS-1/RIP-1 acts as a chaperone to promote 3 to 5 filament growth via highly dynamic engagement with 5 filament ends. Inhibiting ATPase or mutation in RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.

biophysics↗

bioRxiv2020

Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

Mechanistic understanding of biochemical reactions requires structural and kinetic characterization of the underlying chemical processes. However, no single experimental technique can provide this information in a broadly applicable manner and thus structural studies of static macromolecules are often complemented by biophysical analysis. Moreover, the common strategy of utilizing mutants or crosslinking probes to stabilize otherwise short-lived reaction intermediates is prone to trapping off-pathway artefacts and precludes determining the order of molecular events. To overcome these limitations and allow visualisation of biochemical processes at near-atomic spatial resolution and millisecond time scales, we developed a time-resolved sample preparation method for cryo-electron microscopy (trEM). We integrated a modular microfluidic device, featuring a 3D-mixing unit and a delay line of variable length, with a gas-assisted nozzle and motorised plunge-freeze set-up that enables automated, fast, and blot-free sample vitrification. This sample preparation not only preserves high-resolution structural detail but also substantially improves protein distribution across the vitreous ice. We validated the method by examining the formation of RecA filaments on single-stranded DNA. We could reliably visualise reaction intermediates of early filament growth across three orders of magnitude on sub-second timescales. Quantification of the trEM data allowed us to characterize the kinetics of RecA filament growth. The trEM method reported here is versatile, easy to reproduce and thus readily adaptable to a broad spectrum of fundamental questions in biology.

biochemistry↗

bioRxiv2018

CSNAP, the smallest CSN subunit, modulates proteostasis through cullin-RING ubiquitin ligases

The cullin-RING ubiquitin E3 ligase (CRL) family consists of ~250 complexes that catalyze ubiquitylation of proteins to achieve cellular regulation. All CRLs are inhibited by the COP9 signalosome complex (CSN) through both enzymatic (deneddylation) and non-enzymatic (steric) mechanisms. The relative contribution of these two mechanisms is unclear. Here, we decouple the mechanisms using CSNAP, the recently discovered ninth subunit of the CSN. We find that CSNAP reduces the affinity of CSN toward CRL complexes. Removing CSNAP does not affect deneddylation, but leads to global effects on the CRL, causing altered reproductive capacity, suppressed DNA damage response, decreased viability, and delayed cell cycle progression. Thus, although CSNAP is only 2% of the CSN mass, it plays a critical role in the steric regulation of CRLs by the CSN.

biochemistry↗