Biology subjects

Makukhin, N.

Publications and source records attributed to Makukhin, N..

3 recordsLinked to original sources

bioRxiv2026

Photocrosslinking Activity-Based Probes to Capture the Dynamics of Ubiquitin RING E3 Ligase Interactions

Almost all cellular processes are influenced by ubiquitination. A large family of enzymes known as E3 ligases provide the specificity for ubiquitination, with the largest class among them, the RING E3s, comprising over 600 members in humans. RING E3s facilitate transfer of ubiquitin to substrates by constraining the highly dynamic E2-Ub thioester linkage to be primed for attack from the substrate nucleophile. We have established a workflow that uses a modified ubiquitin carrying a photoactivatable crosslinker that once stably linked to the active site of an E2, creates an activity based probe (ABP) to monitor interactions with E3 ligases. Using this, regions of interaction between ubiquitin and a selection of different RING E3 were determined, which not only confirmed existing structures of E2-Ub-RING-E3 complexes but was also used to assess new Ub-E2-E3 models generated in absence of existing structures.

molecular biology↗

bioRxiv2024

Mechanism of degrader-targeted protein ubiquitinability

Small molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and the potential to tackle previously undrugged targets. Thermodynamically stable and kinetically long-lived degrader-mediated ternary complexes can drive faster, more profound and durable target degradation, however the mechanistic features by which they impact on target ubiquitination remain elusive. Here, we solve cryo-EM structures of the VHL Cullin 2 RING E3 ligase complexed with degrader MZ1, target protein Brd4BD2 and primed for catalysis with its cognate E2-ubiquitin bound. We find that Brd4BD2 adopts a favourable orientation towards the E2 active site. In vitro ubiquitination coupled with mass spectrometry illuminates a patch of ubiquitinable lysines on one face of Brd4BD2, with Lys456 showing optimal distance and geometry for nucleophilic attack. Our results demonstrate the proficiency of MZ1 in directing the substrate towards catalysis, explains the favourability of Brd4BD2 for ubiquitination, and reveals the flexibility of the enzyme in capturing sub-optimal lysines. We propose a model for ubiquitinability of degrader-recruited targets that provides a mechanistic blueprint for further rational drug design and optimization. One-Sentence SummaryStructural assembly a PROTAC-mediated complex of whole Cullin RING E3 ligase with bound target and E2-ubiquitin reveals structural and mechanistic insights of specificity for target protein ubiquitination.

biochemistry↗

bioRxiv2018

Structural insights into substrate recognition by the SOCS2 E3 ubiquitin ligase

The suppressor of cytokine signaling 2 (SOCS2) acts as substrate recognition subunit of a Cullin5 E3 ubiquitin ligase complex. SOCS2 binds to phosphotyrosine-modified epitopes as degrons for ubiquitination and proteasomal degradation, yet the molecular basis of substrate recognition has remained elusive. We solved cocrystal structures of SOCS2-ElonginB-ElonginC in complex with phosphorylated peptides from substrates growth hormone receptor (GHR-pY595) and erythropoietin receptor (EpoR-pY426) at 1.98 A{square} and 2.69 A{square}, respectively. Both peptides bind in an extended conformation recapitulating the canonical SH2 domain-pY pose, yet capture different conformations of the EF loop via specific hydrophobic interactions. The flexible BG loop, for the first time fully defined in the electron density, does not contact the substrate degrons directly. Cancer-associated SNPs located around the pY pocket weaken substrate-binding affinity in biophysical assays. Our findings reveal insights into substrate recognition and specificity by SOCS2, and provide a blueprint for small molecule ligand design.

biochemistry↗