SUMMARY In the largest E3 ligase subfamily, Cul3 binds a BTB domain, and an associated protein-interaction domain such as MATH recruits substrates for ubiquitination. Here we present biochemical and structural analyses of the MATH-BTB protein, SPOP. We define a SPOP-binding consensus (SBC), and determine structures revealing recognition of SBCs from the phosphatase Puc, the transcriptional regulator Ci, and the chromatin component MacroH2A. We identify a dimeric SPOP-Cul3 assembly involving a conserved helical structure C-terminal of BTB domains, which we call “3-box” due to its facilitating Cul3-binding and its resemblance to F-/SOCS-boxes in other cullin-based E3s. Structural flexibility between the substrate-binding MATH and Cul3-binding BTB/3-box domains potentially allows a SPOP dimer to engage multiple SBCs found within a single substrate, such as Puc. These studies provide a molecular understanding of how MATH-BTB proteins recruit substrates to Cul3, and how their dimerization and conformational variability may facilitate avid interactions with diverse substrates.

Proteolysis-targeting chimeras (PROTACs) and related molecules that induce targeted protein degradation by the ubiquitin-proteasome system represent a new therapeutic modality and are the focus of great interest, owing to potential advantages over traditional occupancy-based inhibitors with respect to dosing, side effects, drug resistance and modulating 'undruggable' targets. However, the technology is still maturing, and the design elements for successful PROTAC-based drugs are currently being elucidated. Importantly, fewer than 10 of the more than 600 E3 ubiquitin ligases have so far been exploited for targeted protein degradation, and expansion of knowledge in this area is a key opportunity. Here, we briefly discuss lessons learned about targeted protein degradation in chemical biology and drug discovery and systematically review the expression profile, domain architecture and chemical tractability of human E3 ligases that could expand the toolbox for PROTAC discovery. Recent crystal structures of ternary complexes have advanced the understanding of the structural mechanism of PROTACs. Structural studies on VHL and CRBN show that these Cullin-RING E3 ligases (CRLs) form large, modular, U-shaped complexes in which adaptor proteins mediate the interaction between the substrate-binding element (VHL or CRBN) and Cullin scaffolds that bind the RING-domain protein RBX1, leading to the recruitment of a ubiquitinconjugated E2 (refs 9,46,47,48,49,50,51,52) (Fig. 2a,b). The U shape leads to proximal positioning of the E2 and substrate proteins, allowing targeted ubiquitin transfer. The architecture of these large complexes is expected to provide an extended ubiquitylation radius that can accommodate multiple ubiquitylation sites on substrates with diverse sizes and shapes 47,52. In particular, given

Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.

The small molecule thioflavin T (ThT) is a defining probe for the identification and mechanistic study of amyloid fiber formation. As such, ThT is fundamental to investigations of serious diseases such as Alzheimer's disease, Parkinson disease, and type II diabetes. For each disease, a different protein undergoes conformational conversion to a β-sheet rich fiber. The fluorescence of ThT exhibits an increase in quantum yield upon binding these fibers. Despite its widespread use, the structural basis for binding specificity and for the changes to the photophysical properties of ThT remain poorly understood. Here, we report the co-crystal structures of ThT with two alternative states of β-2 microglobulin (β2m); one monomeric, the other an amyloid-like oligomer. In the latter, the dye intercalates between β-sheets orthogonal to the β-strands. Importantly, the fluorophore is bound in such a manner that a photophysically relevant torsion is limited to a range of angles generally associated with low, not high, quantum yield. Quantum mechanical assessment of the fluorophore shows the electronic distribution to be strongly stabilized by aromatic interactions with the protein. Monomeric β2m gives little increase in ThT fluorescence despite showing three fluorophores, at two binding sites, in configurations generally associated with high quantum yield. Our efforts fundamentally extend existing understanding about the origins of amyloid-induced photophysical changes. Specifically, the β-sheet interface that characterizes amyloid acts both sterically and electronically to stabilize the fluorophore's ground state electronic distribution. By preventing the fluorophore from adopting its preferred excited state configuration, nonradiative relaxation pathways are minimized and quantum yield is increased.Alzheimer's | beta-2 microglobulin | Parkinson | TICT | photophysics T he conversion of a normally soluble protein into fibrillar aggregates is of central importance to a range of diseases including Alzheimer's disease, Parkinson disease, and type II diabetes (1). Despite the involvement of distinct proteins in each of these conditions, the fibers formed share a common set of structural and biophysical properties, which define them as amyloid. Amyloid fibers are homopolymeric, noncovalent assemblies, which present morphologically as twisted sets of unbranched filaments. These filaments are composed of two or more β-sheets whose strands run in a direction orthogonal to the long axis of the fiber (2, 3). This arrangement yields a characteristic cross-β pattern by X-ray diffraction, the presence of which represents the gold standard for unequivocal identification of amyloid structure (4, 5).As the routine use of diffraction has proven impractical for most mechanistic studies of amyloid assembly, a considerable fraction of the publication record relies in whole or in part on the use of benzothiazole dyes, most commonly thioflavin T (ThT) (Fig. 1A). The breadth of their use includes histological, kinetic, imaging, and structural studie...

The AMP-activated protein kinase (AMPK) is a potential therapeutic target for metabolic diseases based on its reported actions in the liver and skeletal muscle. We evaluated two distinct direct activators of AMPK: a non-selective activator of all AMPK complexes, PF-739, and an activator selective for AMPK β1-containing complexes, PF-249. In cells and animals, both compounds were effective at activating AMPK in hepatocytes, but only PF-739 was capable of activating AMPK in skeletal muscle. In diabetic mice, PF-739, but not PF-249, caused a rapid lowering of plasma glucose levels that was diminished in the absence of skeletal muscle, but not liver, AMPK heterotrimers and was the result of an increase in systemic glucose disposal with no impact on hepatic glucose production. Studies of PF-739 in cynomolgus monkeys confirmed translation of the glucose lowering and established activation of AMPK in skeletal muscle as a potential therapeutic approach to treat diabetic patients.

AMP-activated protein kinase (AMPK) is a principal metabolic regulator affecting growth and response to cellular stress. Comprised of catalytic and regulatory subunits, each present in multiple forms, AMPK is best described as a family of related enzymes. In recent years, AMPK has emerged as a desirable target for modulation of numerous diseases, yet clinical therapies remain elusive. Challenges result, in part, from an incomplete understanding of the structure and function of full-length heterotrimeric complexes. In this work, we provide the full-length structure of the widely expressed α1β1γ1 isoform of mammalian AMPK, along with detailed kinetic and biophysical characterization. We characterize binding of the broadly studied synthetic activator A769662 and its analogs. Our studies follow on the heels of the recent disclosure of the α2β1γ1 structure and provide insight into the distinct molecular mechanisms of AMPK regulation by AMP and A769662.

SUMMARY Hypoxic stress and hypoxia-inducible factors (HIFs) play important roles in a wide range of tumors. We demonstrate that SPOP, which encodes an E3 ubiquitin ligase component, is a direct transcriptional target of HIFs in clear cell renal cell carcinoma (ccRCC). Furthermore, hypoxia results in cytoplasmic accumulation of SPOP which is sufficient to induce tumorigenesis. This tumorigenic activity occurs through the ubiquitination and degradation of multiple regulators of cellular proliferation and apoptosis, including the tumor suppressor PTEN, ERK phosphatases, the pro-apoptotic molecule Daxx and the Hedgehog pathway transcription factor Gli2. Knockdown of SPOP specifically kills ccRCC cells, indicating that it may be a promising therapeutic target. Collectively, our results indicate that SPOP serves as a regulatory hub to promote ccRCC tumorigenesis.

β-2 microglobulin (β2m) is a globular protein that self-associates into fibrillar amyloid deposits in patients undergoing hemodialysis therapy. Formation of these β-sheet–rich assemblies is a fundamental property of polypeptides that can be triggered by diverse conditions. For β2m, oligomerization into pre-amyloidogenic states occurs in specific response to coordination by Cu2+. Here we report the basis for this self-association at atomic resolution. Metal is not a direct participant in the molecular interface. Rather, binding results in distal alterations enabling the formation of two new surfaces. These interact to form a closed hexameric species. The origins of this include isomerization of a buried and conserved cis-proline previously implicated in the β2m aggregation pathway. The consequences of this isomerization are evident and reveal a molecular basis for the conversion of this robust monomeric protein into an amyloid-competent state.