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Instruments / Technical Area	Publication Details	Published
Krios I-Titan Krios I at Diamond	Mechanism of degrader-targeted protein ubiquitinability Charlotte Crowe , Mark A. Nakasone , Sarah Chandler , Conner Craigon , Gajanan Sathe , Michael H. Tatham , Nikolai Makukhin , Ronald T. Hay , Alessio Ciulli Science Advances 10 DOI: 10.1126/sciadv.ado6492 Diamond Proposal Number(s): [31827] Open Access Show Abstract ▼ Abstract: Small-molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and potential to tackle previously undrugged targets. Stable and long-lived degrader-mediated ternary complexes drive fast and profound target degradation; however, the mechanisms by which they affect target ubiquitination remain elusive. Here, we show cryo-EM structures of the VHL Cullin 2 RING E3 ligase with the degrader MZ1 directing target protein Brd4BD2 toward UBE2R1-ubiquitin, and Lys456 at optimal positioning for nucleophilic attack. In vitro ubiquitination and mass spectrometry illuminate a patch of favorably ubiquitinable lysines on one face of Brd4BD2, with cellular degradation and ubiquitinomics confirming the importance of Lys456 and nearby Lys368/Lys445, identifying the “ubiquitination zone.” Our results demonstrate the proficiency of MZ1 in positioning the substrate for catalysis, the favorability of Brd4BD2 for ubiquitination by UBE2R1, and the flexibility of CRL2 for capturing suboptimal lysines. We propose a model for ubiquitinability of degrader-recruited targets, providing a mechanistic blueprint for further rational drug design.	Oct 2024
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2 Sarath Ramachandran , Nikolai Makukhin , Kevin Haubrich , Manjula Nagala , Beth Forrester , Dylan M. Lynch , Ryan Casement , Andrea Testa , Elvira Bruno , Rosaria Gitto , Alessio Ciulli Nature Communications 14 DOI: 10.1038/s41467-023-41894-3 Open Access Show Abstract ▼ Abstract: The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation.	Oct 2023
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural insights into substrate recognition by the SOCS2 E3 ubiquitin ligase Wei-Wei Kung , Sarath Ramachandran , Nikolai Makukhin , Elvira Bruno , Alessio Ciulli Nature Communications 10, 2534 DOI: 10.1038/s41467-019-10190-4 Diamond Proposal Number(s): [14980] Open Access Show Abstract ▼ Abstract: 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. Here, we report co-crystal 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 Å and 2.69 Å, respectively. Both peptides bind in an extended conformation recapitulating the canonical SH2 domain-pY pose, but capture different conformations of the EF loop via specific hydrophobic interactions. The flexible BG loop is fully defined in the electron density, and does not contact the substrate degron 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.	Jun 2019

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