CRISPR-suppressor scanning reveals a nonenzymatic role of LSD1 in AML.

Understanding the mechanism of small molecules is a critical challenge in chemical biology and drug discovery. Medicinal chemistry is essential for elucidating drug mechanism, enabling variation of small molecule structure to gain structure-activity relationships (SARs). However, the development of complementary approaches that systematically vary target protein structure could provide equally informative SARs for investigating drug mechanism and protein function. Here we explore the ability of CRISPR-Cas9 mutagenesis to profile the interactions between lysine-specific histone demethylase 1 (LSD1) and chemical inhibitors in the context of acute myeloid leukemia (AML). Through this approach, termed CRISPR-suppressor scanning, we elucidate drug mechanism of action by showing that LSD1 enzyme activity is not required for AML survival and that LSD1 inhibitors instead function by disrupting interactions between LSD1 and the transcription factor GFI1B on chromatin. Our studies clarify how LSD1 inhibitors mechanistically operate in AML and demonstrate how CRISPR-suppressor scanning can uncover novel aspects of target biology.

Zinc-binding structure of a catalytic amyloid from solid-state NMR.

Throughout biology, amyloids are key structures in both functional proteins and the end product of pathologic protein misfolding. Amyloids might also represent an early precursor in the evolution of life because of their small molecular size and their ability to self-purify and catalyze chemical reactions. They also provide attractive backbones for advanced materials. When ß-strands of an amyloid are arranged parallel and in register, side chains from the same position of each chain align, facilitating metal chelation when the residues are good ligands such as histidine. High-resolution structures of metalloamyloids are needed to understand the molecular bases of metal-amyloid interactions. Here we combine solid-state NMR and structural bioinformatics to determine the structure of a zinc-bound metalloamyloid that catalyzes ester hydrolysis. The peptide forms amphiphilic parallel ß-sheets that assemble into stacked bilayers with alternating hydrophobic and polar interfaces. The hydrophobic interface is stabilized by apolar side chains from adjacent sheets, whereas the hydrated polar interface houses the Zn2+-binding histidines with binding geometries unusual in proteins. Each Zn2+ has two bis-coordinated histidine ligands, which bridge adjacent strands to form an infinite metal-ligand chain along the fibril axis. A third histidine completes the protein ligand environment, leaving a free site on the Zn2+ for water activation. This structure defines a class of materials, which we call metal-peptide frameworks. The structure reveals a delicate interplay through which metal ions stabilize the amyloid structure, which in turn shapes the ligand geometry and catalytic reactivity of Zn2.

A Designed Enzyme Promotes Selective Post-translational Acylation.

A computationally designed, allosterically regulated catalyst (CaM M144H) produced by substituting a single residue in calmodulin, a non-enzymatic protein, is capable of efficient and site selective post-translational acylation of lysines in peptides with highly diverse sequences. Calmodulin's binding partners are involved in regulating a large number of cellular processes; this new chemical-biology tool will help to identify them and provide structural insight into their interactions with calmodulin.

Short Self-Assembling Peptides Are Able to Bind to Copper and Activate Oxygen.

We have shown that de novo designed peptides self-assemble in the presence of copper to create supramolecular assemblies capable of carrying out the oxidation of dimethoxyphenol in the presence of dioxygen. Formation of the supramolecular assembly, which is akin to a protein fold, is critical for productive catalysis since peptides possessing the same functional groups but lacking the ability to self-assemble do not catalyze substrate oxidation. The ease with which we have discovered robust and productive oxygen activation catalysts suggests that these prion-like assemblies might have served as intermediates in the evolution of enzymatic function and opens the path for the development of new catalyst nanomaterials.

New Tricks for Old Proteins: Single Mutations in a Nonenzymatic Protein Give Rise to Various Enzymatic Activities.

Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution.

Functional characterization of a melittin analog containing a non-natural tryptophan analog.

Tryptophan (Trp) is a naturally occurring amino acid, which exhibits fluorescence emission properties that are dependent on the polarity of the local environment around the Trp side chain. However, this sensitivity also complicates interpretation of fluorescence emission data. A non-natural analogue of tryptophan, ß-(1-azulenyl)-L-alanine, exhibits fluorescence insensitive to local solvent polarity and does not impact the structure or characteristics of several peptides examined. In this study, we investigated the effect of replacing Trp with ß-(1-azulenyl)-L-alanine in the well-known bee-venom peptide melittin. This peptide provides a model framework for investigating the impact of replacing Trp with ß-(1-azulenyl)-L-alanine in a functional peptide system that undergoes significant shifts in Trp fluorescence emission upon binding to lipid bilayers. Microbiological methods including assessment of the antimicrobial activity by minimal inhibitory concentration (MIC) assays and bacterial membrane permeability assays indicated little difference between the Trp and the ß-(1-azulenyl)-L-alanine-substituted versions of melittin. Circular dichroism spectroscopy showed both that peptides adopted the expected α-helical structures when bound to phospholipid bilayers and electrophysiological analysis indicated that both created membrane disruptions leading to significant conductance increases across model membranes. Both peptides exhibited a marked protection of the respective fluorophores when bound to bilayers indicating a similar membrane-bound topology. As expected, while fluorescence quenching and CD indicate the peptides are stably bound to lipid vesicles, the peptide containing ß-(1-azulenyl)-L-alanine exhibited no fluorescence emission shift upon binding while the natural Trp exhibited >10 nm shift in emission spectrum barycenter. Taken together, the ß-(1-azulenyl)-L-alanine can serve as a solvent insensitive alternative to Trp that does not have significant impacts on structure or function of membrane interacting peptides.

KBTBD4 Cancer Hotspot Mutations Drive Neomorphic Degradation of HDAC1/2 Corepressor Complexes.

Cancer mutations can create neomorphic protein-protein interactions to drive aberrant function 1 . As a substrate receptor of the CULLIN3-RBX1 E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma (MB) 2 , the most common embryonal brain tumor in children, and pineoblastoma 3 . These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST 4 . However, their mechanism of action remains unresolved. Here, we elucidate the mechanistic basis by which KBTBD4 mutations promote CoREST degradation through engaging HDAC1/2, the direct neomorphic target of the substrate receptor. Using deep mutational scanning, we systematically map the mutational landscape of the KBTBD4 cancer hotspot, revealing distinct preferences by which insertions and substitutions can promote gain-of-function and the critical residues involved in the hotspot interaction. Cryo-electron microscopy (cryo-EM) analysis of two distinct KBTBD4 cancer mutants bound to LSD1-HDAC1-CoREST reveals that a KBTBD4 homodimer asymmetrically engages HDAC1 with two KELCH-repeat propeller domains. The interface between HDAC1 and one of the KBTBD4 propellers is stabilized by the MB mutations, which directly insert a bulky side chain into the active site pocket of HDAC1. Our structural and mutational analyses inform how this hotspot E3-neo-substrate interface can be chemically modulated. First, our results unveil a converging shape complementarity-based mechanism between gain-of-function E3 mutations and a molecular glue degrader, UM171. Second, we demonstrate that HDAC1/2 inhibitors can block the mutant KBTBD4-HDAC1 interface, the aberrant degradation of CoREST, and the growth of KBTBD4-mutant MB models. Altogether, our work reveals the structural and mechanistic basis of cancer mutation-driven neomorphic protein-protein interactions and pharmacological strategies to modulate their action for therapeutic applications.

Asymmetric Engagement of Dimeric CRL3 KBTBD4 by the Molecular Glue UM171 Licenses Degradation of HDAC1/2 Complexes.

UM171 is a potent small molecule agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal, a process that is tightly controlled by epigenetic regulation. By co-opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, UM171 promotes the degradation of members of the CoREST transcriptional corepressor complex, thereby limiting HSC attrition. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1 to promote the degradation of select HDAC1/2 corepressor complexes. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy (cryo-EM) analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an unexpected asymmetric assembly, in which a single UM171 molecule enables a pair of KBTBD4 KELCH-repeat propeller domains to recruit HDAC1 by clamping on its catalytic domain. One of the KBTBD4 propellers partially masks the rim of the HDAC1 active site pocket, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a separate and distinct interface. The overall neomorphic interaction is further buttressed by an endogenous cofactor of HDAC1-CoREST, inositol hexakisphosphate, which makes direct contacts with KBTBD4 and acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces defined by cryo-EM is demonstrated by in situ base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, our results reveal how the cooperativity offered by a large dimeric CRL E3 family can be leveraged by a small molecule degrader and establish for the first time a dual molecular glue paradigm.

Activity-based CRISPR scanning uncovers allostery in DNA methylation maintenance machinery.

Allostery enables dynamic control of protein function. A paradigmatic example is the tightly orchestrated process of DNA methylation maintenance. Despite the fundamental importance of allosteric sites, their identification remains highly challenging. Here, we perform CRISPR scanning on the essential maintenance methylation machinery-DNMT1 and its partner UHRF1-with the activity-based inhibitor decitabine to uncover allosteric mechanisms regulating DNMT1. In contrast to non-covalent DNMT1 inhibition, activity-based selection implicates numerous regions outside the catalytic domain in DNMT1 function. Through computational analyses, we identify putative mutational hotspots in DNMT1 distal from the active site that encompass mutations spanning a multi-domain autoinhibitory interface and the uncharacterized BAH2 domain. We biochemically characterize these mutations as gain-of-function, exhibiting increased DNMT1 activity. Extrapolating our analysis to UHRF1, we discern putative gain-of-function mutations in multiple domains, including key residues across the autoinhibitory TTD-PBR interface. Collectively, our study highlights the utility of activity-based CRISPR scanning for nominating candidate allosteric sites, and more broadly, introduces new analytical tools that further refine the CRISPR scanning framework.

Profiling the Landscape of Drug Resistance Mutations in Neosubstrates to Molecular Glue Degraders.

Targeted protein degradation (TPD) holds immense promise for drug discovery, but mechanisms of acquired resistance to degraders remain to be fully identified. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)-suppressor scanning to identify mechanistic classes of drug resistance mutations to molecular glue degraders in GSPT1 and RBM39, neosubstrates targeted by E3 ligase substrate receptors cereblon and DCAF15, respectively. While many mutations directly alter the ternary complex heterodimerization surface, distal resistance sites were also identified. Several distal mutations in RBM39 led to modest decreases in degradation, yet can enable cell survival, underscoring how small differences in degradation can lead to resistance. Integrative analysis of resistance sites across GSPT1 and RBM39 revealed varying levels of sequence conservation and mutational constraint that control the emergence of different resistance mechanisms, highlighting that many regions co-opted by TPD are nonessential. Altogether, our study identifies common resistance mechanisms for molecular glue degraders and outlines a general approach to survey neosubstrate requirements necessary for effective degradation.

Minimalist IR and fluorescence probes of protein function.

Spectroscopic studies of small proteins and peptides, especially those requiring fine spatial and/or temporal resolution, demand synthetic probes that confer the minimal possible steric and functional change on the native properties. Here we review the recent progress in development of minimally disruptive probes for fluorescence and infrared spectroscopies, as well as the methods to efficiently incorporate them into proteins. Advances in spectroscopy on the one hand result in high specialization of synthetic probes for a particular purpose, but on the other hand allow for the same probes be used for different techniques to gather complementary biochemical information.

ß-(1-Azulenyl)-L-alanine--a functional probe for determination of pKa of histidine residues.

ß-(1-Azulenyl)-L-alanine (AzAla) can be incorporated into the influenza A virus M2 proton channel. AzAla's sensitivity to the protonation state of the nearby histidines and the lack of environmental fluorescence dependence allow for direct and straightforward determination of histidine pKa values in ion channels.