Two diphosphorylated degrons control c-Myc degradation by the Fbw7 tumor suppressor.

c-Myc (hereafter, Myc) is a cancer driver whose abundance is regulated by the SCFFbw7 ubiquitin ligase and proteasomal degradation. Fbw7 binds to a phosphorylated Myc degron centered at threonine 58 (T58), and mutations of Fbw7 or T58 impair Myc degradation in cancers. Here, we identify a second Fbw7 phosphodegron at Myc T244 that is required for Myc ubiquitylation and acts in concert with T58 to engage Fbw7. While Ras-dependent Myc serine 62 phosphorylation (pS62) is thought to stabilize Myc by preventing Fbw7 binding, we find instead that pS62 greatly enhances Fbw7 binding and is an integral part of a high-affinity degron. Crystallographic studies revealed that both degrons bind Fbw7 in their diphosphorylated forms and that the T244 degron is recognized via a unique mode involving Fbw7 arginine 689 (R689), a mutational hotspot in cancers. These insights have important implications for Myc-associated tumorigenesis and therapeutic strategies targeting Myc stability.

The structure of the cysteine-rich region from human histone-lysine N-methyltransferase EHMT2 (G9a).

Euchromatic histone-lysine N-methyltransferase 1 (EHMT1; G9a-like protein; GLP) and euchromatic histone-lysine N-methyltransferase 2 (EHMT2; G9a) are protein lysine methyltransferases that regulate gene expression and are essential for development and the ability of organisms to change and adapt. In addition to ankyrin repeats and the catalytic SET domain, the EHMT proteins contain a unique cysteine-rich region (CRR) that mediates protein-protein interactions and recruitment of the methyltransferases to specific sites in chromatin. We have determined the structure of the CRR from human EHMT2 by X-ray crystallography and show that the CRR adopts an unusual compact fold with four bound zinc atoms. The structure consists of a RING domain preceded by a smaller zinc-binding motif and an N-terminal segment. The smaller zinc-binding motif straddles the N-terminal end of the RING domain, and the N-terminal segment runs in an extended conformation along one side of the structure and interacts with both the smaller zinc-binding motif and the RING domain. The interface between the N-terminal segment and the RING domain includes one of the zinc atoms. The RING domain is partially sequestered within the CRR and unlikely to function as a ubiquitin ligase.

Degronomics: Mapping the Interacting Peptidome of a Ubiquitin Ligase Using an Integrative Mass Spectrometry Strategy.

Human cells make use of hundreds of unique ubiquitin E3 ligases to ensure proteome fidelity and control cellular functions by promoting protein degradation. These processes require exquisite selectivity, but the individual roles of most E3s remain poorly characterized in part due to the challenges associated with identifying, quantifying, and validating substrates for each E3. We report an integrative mass spectrometry (MS) strategy for characterizing protein fragments that interact with KLHDC2, a human E3 that recognizes the extreme C-terminus of substrates. Using a combination of native MS, native top-down MS, MS of destabilized samples, and liquid chromatography MS, we identified and quantified a near complete fraction of the KLHDC2-binding peptidome in E. coli cells. This degronome includes peptides that originate from a variety of proteins. Although all identified protein fragments are terminated by diglycine or glycylalanine, the preceding amino acids are diverse. These results significantly expand our understanding of the sequences that can be recognized by KLHDC2, which provides insight into the potential substrates of this E3 in humans. We anticipate that this integrative MS strategy could be leveraged more broadly to characterize the degronomes of other E3 ligase substrate receptors, including those that adhere to the more common N-end rule for substrate recognition. Therefore, this work advances "degronomics," i.e., identifying, quantifying, and validating functional E3:peptide interactions in order to determine the individual roles of each E3.

Recognition of the Diglycine C-End Degron by CRL2KLHDC2 Ubiquitin Ligase.

Aberrant proteins can be deleterious to cells and are cleared by the ubiquitin-proteasome system. A group of C-end degrons that are recognized by specific cullin-RING ubiquitin E3 ligases (CRLs) has recently been identified in some of these abnormal polypeptides. Here, we report three crystal structures of a CRL2 substrate receptor, KLHDC2, in complex with the diglycine-ending C-end degrons of two early-terminated selenoproteins and the N-terminal proteolytic fragment of USP1. The E3 recognizes the degron peptides in a similarly coiled conformation and cradles their C-terminal diglycine with a deep surface pocket. By hydrogen bonding with multiple backbone carbonyls of the peptides, KLHDC2 further locks in the otherwise degenerate degrons with a compact interface and unexpected high affinities. Our results reveal the structural mechanism by which KLHDC2 recognizes the simplest C-end degron and suggest a functional necessity of the E3 to tightly maintain the low abundance of its select substrates.

The Interaction between the Third Type III Domain from Fibronectin and Anastellin Involves ß-Strand Exchange.

Anastellin is a small recombinant fragment derived from the extracellular matrix protein fibronectin; it comprises the first type III (FN3) domain without the two N-terminal ß-strands. It inhibits angiogenesis, tumor growth, and metastasis in mouse models and requires endogenous fibronectin for its in vivo anti-angiogenic activity. It binds to fibronectin in vitro and converts the soluble protein to insoluble fibrils that structurally and functionally resemble fibronectin fibrils deposited in the extracellular matrix by cells. Anastellin binds to several FN3 domains in fibronectin, but how it interacts with these domains and why the interactions lead to aggregation of fibronectin are not well understood. In this work, we investigated the interaction between anastellin and the third FN3 domain (3FN3) from fibronectin. We show that anastellin binds with high affinity to a peptide comprising the two N-terminal ß-strands from 3FN3, and we present here the structure of the resulting complex. The peptide and anastellin form a composite FN3 domain, with the two N-terminal ß-strands from 3FN3 bound in place of the two ß-strands that are missing in anastellin. We also demonstrate using disulfide cross-linking that a similar interaction involving the two N-terminal ß-strands of 3FN3 occurs when intact 3FN3 binds to anastellin. 3FN3 adopts a compact globular fold in solution, and to interact with anastellin in a manner consistent with our data, it has to open up and expose a ß-strand edge that is not accessible in the context of the folded domain.