The energetic and allosteric landscape for KRAS inhibition.

Thousands of proteins have been validated genetically as therapeutic targets for human diseases1. However, very few have been successfully targeted, and many are considered 'undruggable'. This is particularly true for proteins that function via protein-protein interactions-direct inhibition of binding interfaces is difficult and requires the identification of allosteric sites. However, most proteins have no known allosteric sites, and a comprehensive allosteric map does not exist for any protein. Here we address this shortcoming by charting multiple global atlases of inhibitory allosteric communication in KRAS. We quantified the effects of more than 26,000 mutations on the folding of KRAS and its binding to six interaction partners. Genetic interactions in double mutants enabled us to perform biophysical measurements at scale, inferring more than 22,000 causal free energy changes. These energy landscapes quantify how mutations tune the binding specificity of a signalling protein and map the inhibitory allosteric sites for an important therapeutic target. Allosteric propagation is particularly effective across the central ß-sheet of KRAS, and multiple surface pockets are genetically validated as allosterically active, including a distal pocket in the C-terminal lobe of the protein. Allosteric mutations typically inhibit binding to all tested effectors, but they can also change the binding specificity, revealing the regulatory, evolutionary and therapeutic potential to tune pathway activation. Using the approach described here, it should be possible to rapidly and comprehensively identify allosteric target sites in many proteins.

Rational optimization of a transcription factor activation domain inhibitor.

Transcription factors are among the most attractive therapeutic targets but are considered largely 'undruggable' in part due to the intrinsically disordered nature of their activation domains. Here we show that the aromatic character of the activation domain of the androgen receptor, a therapeutic target for castration-resistant prostate cancer, is key for its activity as transcription factor, allowing it to translocate to the nucleus and partition into transcriptional condensates upon activation by androgens. On the basis of our understanding of the interactions stabilizing such condensates and of the structure that the domain adopts upon condensation, we optimized the structure of a small-molecule inhibitor previously identified by phenotypic screening. The optimized compounds had more affinity for their target, inhibited androgen-receptor-dependent transcriptional programs, and had an antitumorigenic effect in models of castration-resistant prostate cancer in cells and in vivo. These results suggest that it is possible to rationally optimize, and potentially even to design, small molecules that target the activation domains of oncogenic transcription factors.

A glutamine-based single α-helix scaffold to target globular proteins.

The binding of intrinsically disordered proteins to globular ones can require the folding of motifs into α-helices. These interactions offer opportunities for therapeutic intervention but their modulation with small molecules is challenging because they bury large surfaces. Linear peptides that display the residues that are key for binding can be targeted to globular proteins when they form stable helices, which in most cases requires their chemical modification. Here we present rules to design peptides that fold into single α-helices by instead concatenating glutamine side chain to main chain hydrogen bonds recently discovered in polyglutamine helices. The resulting peptides are uncharged, contain only natural amino acids, and their sequences can be optimized to interact with specific targets. Our results provide design rules to obtain single α-helices for a wide range of applications in protein engineering and drug design.

An Adhesive Peptide from the C-Terminal Domain of α-Synuclein for Single-Layer Adsorption of Nanoparticles onto Substrates.

The two-dimensional (2D) homogeneous assembly of nanoparticle monolayer arrays onto a broad range of substrates constitutes an important challenge for chemistry, nanotechnology, and material science. α-Synuclein (αS) is an intrinsically disordered protein associated with neuronal protein complexes and has a high degree of structural plasticity and chaperone activity. The C-terminal domain of αS has been linked to the noncovalent interactions of this protein with biological targets and the activity of αS in presynaptic connections. Herein, we have systematically studied peptide fragments of the chaperone-active C-terminal sequence of αS and identified a 17-residue peptide that preserves the versatile binding nature of αS. Attachment of this short peptide to gold nanoparticles afforded colloidally stable nanoparticle suspensions that allowed the homogeneous 2D adhesion of the conjugates onto a wide variety of surfaces, including the formation of crystalline nanoparticle superlattices. The peptide sequence and the strategy reported here describe a new adhesive molecule for the controlled monolayer adhesion of metal nanoparticles and sets a stepping-stone toward the potential application of the adhesive properties of αS.

Recombinant Production of Monomeric Isotope-Enriched Aggregation-Prone Peptides: Polyglutamine Tracts and Beyond.

High solvent exposure of certain sequences located in intrinsically disordered regions (IDRs) may eventually lead to aggregation, as is the case for some low-complexity regions (LCRs) and short linear motifs (SLiMs). In particular, polyglutamine (polyQ) tracts are LCRs of variable length highly enriched in glutamine residues. They are common in transcription factors, and their length can have an impact on transcriptional activity. In nine proteins, polyQ tract expansions beyond specific thresholds cause nine neurodegenerative diseases, and aggregates formed by the protein harboring the polyQ tract can be detected in affected individuals. A structural characterization of polyQ proteins in their monomeric form is key to understand how their expansion can affect their aggregation propensity. In this regard, nuclear magnetic resonance (NMR) spectroscopy can provide high-resolution structural information. Here, we present a protocol to prepare monomeric samples of isotope-enriched short helical polyQ peptides based on the sequence of the androgen receptor (AR) suitable for NMR characterization and suggest different ways to adapt it for the production and monomerization of other relatively short IDR sequences and SLiMs.

Side chain to main chain hydrogen bonds stabilize a polyglutamine helix in a transcription factor.

Polyglutamine (polyQ) tracts are regions of low sequence complexity frequently found in transcription factors. Tract length often correlates with transcriptional activity and expansion beyond specific thresholds in certain human proteins is the cause of polyQ disorders. To study the structural basis of the association between tract length, transcriptional activity and disease, we addressed how the conformation of the polyQ tract of the androgen receptor, associated with spinobulbar muscular atrophy (SBMA), depends on its length. Here we report that this sequence folds into a helical structure stabilized by unconventional hydrogen bonds between glutamine side chains and main chain carbonyl groups, and that its helicity directly correlates with tract length. These unusual hydrogen bonds are bifurcate with the conventional hydrogen bonds stabilizing α-helices. Our findings suggest a plausible rationale for the association between polyQ tract length and androgen receptor transcriptional activity and have implications for establishing the mechanistic basis of SBMA.

EPI-001, A Compound Active against Castration-Resistant Prostate Cancer, Targets Transactivation Unit 5 of the Androgen Receptor.

Castration-resistant prostate cancer is the lethal condition suffered by prostate cancer patients that become refractory to androgen deprivation therapy. EPI-001 is a recently identified compound active against this condition that modulates the activity of the androgen receptor, a nuclear receptor that is essential for disease progression. The mechanism by which this compound exerts its inhibitory activity is however not yet fully understood. Here we show, by using high resolution solution nuclear magnetic resonance spectroscopy, that EPI-001 selectively interacts with a partially folded region of the transactivation domain of the androgen receptor, known as transactivation unit 5, that is key for the ability of prostate cells to proliferate in the absence of androgens, a distinctive feature of castration-resistant prostate cancer. Our results can contribute to the development of more potent and less toxic novel androgen receptor antagonists for treating this disease.

Structural basis of the activation and degradation mechanisms of the E3 ubiquitin ligase Nedd4L.

We investigated the mechanisms of activation and degradation of the E3 ubiquitin ligase Nedd4L combining the available biochemical information with complementary biophysical techniques. Using nuclear magnetic resonance spectroscopy, we identified that the C2 domain binds Ca(2+) and inositol 1,4,5-trisphosphate (IP3) using the same interface that is used to interact with the HECT domain. Thus, we propose that the transition from the closed to the active form is regulated by a competition of IP3 and Ca(2+) with the HECT domain for binding to the C2 domain. We performed relaxation experiments and molecular dynamic simulations to determine the flexibility of the HECT structure and observed that its conserved PY motif can become solvent-exposed when the unfolding process is initiated. The structure of the WW3 domain bound to the HECT-PY site reveals the details of this interaction, suggesting a possible auto-ubquitination mechanism using two molecules, a partially unfolded one and a fully functional Nedd4L counterpart.

A Smad action turnover switch operated by WW domain readers of a phosphoserine code.

When directed to the nucleus by TGF-ß or BMP signals, Smad proteins undergo cyclin-dependent kinase 8/9 (CDK8/9) and glycogen synthase kinase-3 (GSK3) phosphorylations that mediate the binding of YAP and Pin1 for transcriptional action, and of ubiquitin ligases Smurf1 and Nedd4L for Smad destruction. Here we demonstrate that there is an order of events-Smad activation first and destruction later-and that it is controlled by a switch in the recognition of Smad phosphoserines by WW domains in their binding partners. In the BMP pathway, Smad1 phosphorylation by CDK8/9 creates binding sites for the WW domains of YAP, and subsequent phosphorylation by GSK3 switches off YAP binding and adds binding sites for Smurf1 WW domains. Similarly, in the TGF-ß pathway, Smad3 phosphorylation by CDK8/9 creates binding sites for Pin1 and GSK3, then adds sites to enhance Nedd4L binding. Thus, a Smad phosphoserine code and a set of WW domain code readers provide an efficient solution to the problem of coupling TGF-ß signal delivery to turnover of the Smad signal transducers.