Targeting the Plasmodium falciparum UCHL3 ubiquitin hydrolase using chemically constrained peptides.

The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt P. falciparum growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of antimalarial therapeutics.

Investigating the Interactions of the Cucumber Mosaic Virus 2b Protein with the Viral 1a Replicase Component and the Cellular RNA Silencing Factor Argonaute 1.

The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein's host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In Arabidopsis thaliana, if 2b-induced inhibition of AGO1 is too efficient, it induces reinforcement of antiviral silencing by AGO2 and triggers increased resistance against aphids, CMV's insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein, which sequesters sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated, and red and green fluorescent protein fusions were used to investigate subcellular colocalization with AGO1 and the 1a protein. The effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b protein's interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b protein's ability to interact with AGO1, the 1a protein, and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.

Influence of point mutations on PR65 conformational adaptability: Insights from molecular simulations and nanoaperture optical tweezers.

PR65 is the HEAT repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem repeat protein. Its conformational mechanics plays a crucial role in PP2A function by opening/closing substrate binding/catalysis interface. Using in silico saturation mutagenesis, we identified PR65 "hinge" residues whose substitutions could alter its conformational adaptability and thereby PP2A function, and selected six mutations that were verified to be expressed and soluble. Molecular simulations and nanoaperture optical tweezers revealed consistent results on the specific effects of the mutations on the structure and dynamics of PR65. Two mutants observed in simulations to stabilize extended/open conformations exhibited higher corner frequencies and lower translational scattering in experiments, indicating a shift toward extended conformations, whereas another displayed the opposite features, confirmed by both simulations and experiments. The study highlights the power of single-molecule nanoaperture-based tweezers integrated with in silico approaches for exploring the effect of mutations on protein structure and dynamics.

Red-light modulated ortho-chloro azobenzene photoswitch for peptide stapling via aromatic substitution.

The application of peptide stapling using photoswitchable linkers has gained notable interest for potential therapeutic applications. However, many existing methodologies of photoswitching still rely on the use of tissue-damaging and weakly skin-penetrating UV light. Herein, we describe the development of a tetra-ortho-chloro azobenzene linker that was successfully used for cysteine-selective peptide stapling via SNAr. This linker facilitates precise photocontrol of peptide structure via trans to cis isomerisation under red light irradiation. As a proof-of-concept, we applied the developed peptide stapling platform to a modified PMI peptide, targeting the inhibition of MDM2/p53 protein-protein interaction (PPI). Biophysical characterisation of the photoswitchable peptide by competitive fluorescence polarisation showed a significant difference in affinity between the trans and cis isomer for the p53-interacting domain of the human MDM2. Remarkably, the cis isomer displayed a >240-fold higher potency. To the best of our knowledge, this is the highest reported difference in binding affinity between isoforms of a photoswitchable therapeutic peptide. Overall, our findings demonstrate the potential of this novel photoswitchable peptide stapling system for tuneable, selective modulation of PPIs via visible-light isomerisation with deeply-tissue penetrating red light.

Tandem-repeat proteins conformational mechanics are optimized to facilitate functional interactions and complexations.

The architectures of tandem-repeat proteins are distinct from those of globular proteins. Individual modules, each comprising small structural motifs of 20-40 residues, are arrayed in a quasi one-dimensional fashion to form striking, elongated, horseshoe-like, and superhelical architectures, stabilized solely by short-range interaction. The spring-like shapes of repeat arrays point to elastic modes of action, and these proteins function as adapter molecules or 'hubs,' propagating signals within multi-subunit assemblies in diverse biological contexts. This flexibility is apparent in the dramatic variability observed in the structures of tandem-repeat proteins in different complexes. Here, using computational analysis, we demonstrate the striking ability of just one or a few global motions to recapitulate these structures. These findings show how the mechanics of repeat arrays are robustly enabled by their unique architecture. Thus, the repeating architecture has been optimized by evolution to favor functional modes of motions. The global motions enabling functional transitions can be fully visualized at http://bahargroup.org/tr_web.

Influence of Point Mutations on PR65 Conformational Adaptability: Insights from Nanoaperture Optical Tweezer Experiments and Molecular Simulations.

PR65 is the HEAT-repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem-repeat protein, forming a spring-like architecture. PR65 conformational mechanics play a crucial role in PP2A function by opening/closing the substrate-binding/catalysis interface. Using in-silico saturation mutagenesis we identified "hinge" residues of PR65, whose substitutions are predicted to restrict its conformational adaptability and thereby disrupt PP2A function. Molecular simulations revealed that a subset of hinge mutations stabilized the extended/open conformation, whereas another had the opposite effect. By trapping in nanoaperture optical tweezer, we characterized PR65 motion and showed that the former mutants exhibited higher corner frequencies and lower translational scattering, indicating a shift towards extended conformations, whereas the latter showed the opposite behavior. Thus, experiments confirm the conformations predicted computationally. The study highlights the utility of nanoaperture-based tweezers for exploring structure and dynamics, and the power of integrating this single-molecule method with in silico approaches.

Interaction between huntingtin exon 1 and HEAT repeat structure probed by chimeric model proteins.

Huntington disease (HD) is associated with aggregation of huntingtin (HTT) protein containing over 35 continuous Q residues within the N-terminal exon 1 encoded region. The C-terminal of the HTT protein consists mainly of HEAT repeat structure which serves as a scaffold for multiple cellular activities. Structural and biochemical analysis of the intact HTT protein has been hampered by its huge size (~300 kDa) and most in vitro studies to date have focused on the properties of the exon 1 region. To explore the interaction between HTT exon 1 and the HEAT repeat structure, we constructed chimeric proteins containing the N-terminal HTT exon 1 region and the HEAT repeat protein PR65/A. The results indicate that HTT exon 1 slightly destabilizes the downstream HEAT repeat structure and endows the HEAT repeat structure with more conformational flexibility. Wild-type and pathological lengths of polyQ did not show differences in the interaction between HTT exon 1 and the HEAT repeats. With the C-terminal fusion of PR65/A, HTT exon 1 containing pathological lengths of polyQ could still form amyloid fibrils, but the higher-order architecture of fibrils and kinetics of fibril formation were affected by the C-terminal fusion of HEAT repeats. This indicates that interaction between HTT exon 1 and HEAT repeat structure is compatible with both normal function of HTT protein and the pathogenesis of HD, and this study provides a potential model for further exploration.

Cooperative mechanics of PR65 scaffold underlies the allosteric regulation of the phosphatase PP2A.

PR65, a horseshoe-shaped scaffold composed of 15 HEAT (observed in Huntingtin, elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) repeats, forms, together with catalytic and regulatory subunits, the heterotrimeric protein phosphatase PP2A. We examined the role of PR65 in enabling PP2A enzymatic activity with computations at various levels of complexity, including hybrid approaches that combine full-atomic and elastic network models. Our study points to the high flexibility of this scaffold allowing for end-to-end distance fluctuations of 40-50 Å between compact and extended conformations. Notably, the intrinsic dynamics of PR65 facilitates complexation with the catalytic subunit and is retained in the PP2A complex enabling PR65 to engage the two domains of the catalytic subunit and provide the mechanical framework for enzymatic activity, with support from the regulatory subunit. In particular, the intra-repeat coils at the C-terminal arm play an important role in allosterically mediating the collective dynamics of PP2A, pointing to target sites for modulating PR65 function.

Counting Degrons: Lessons From Multivalent Substrates for Targeted Protein Degradation.

E3s comprise a structurally diverse group of at least 800 members, most of which target multiple substrates through specific and regulated protein-protein interactions. These interactions typically rely on short linear motifs (SLiMs), called "degrons", in an intrinsically disordered region (IDR) of the substrate, with variable rules of engagement governing different E3-docking events. These rules of engagement are of importance to the field of targeted protein degradation (TPD), where substrate ubiquitination and destruction require tools to effectively harness ubiquitin ligases (E3s). Substrates are often found to contain multiple degrons, or multiple copies of a degron, contributing to the affinity and selectivity of the substrate for its E3. One important paradigm for E3-substrate docking is presented by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit E3 ligase that targets hundreds of proteins for destruction during mitotic exit. APC/C substrate targeting takes place in an ordered manner thought to depend on tightly regulated interactions of substrates, with docking sites provided by the substoichiometric APC/C substrate adaptors and coactivators, Cdc20 or Cdh1/FZR1. Both structural and functional studies of individual APC/C substrates indicate that productive ubiquitination usually requires more than one degron, and that degrons are of different types docking to distinct sites on the coactivators. However, the dynamic nature of APC/C substrate recruitment, and the influence of multiple degrons, remains poorly understood. Here we review the significance of multiple degrons in a number of E3-substrate interactions that have been studied in detail, illustrating distinct kinetic effects of multivalency and allovalency, before addressing the role of multiple degrons in APC/C substrates, key to understanding ordered substrate destruction by APC/C. Lastly, we consider how lessons learnt from these studies can be applied in the design of TPD tools.

Multivalent Interaction of Beta-Catenin With its Intrinsically Disordered Binding Partner Adenomatous Polyposis Coli.

The Wnt signalling pathway plays key roles in cell proliferation, differentiation and fate decisions in embryonic development and maintenance of adult tissues, and the twelve Armadillo (ARM) repeat-containing protein ß-catenin acts as the signal transducer in this pathway. Here we investigate the interaction between ß-catenin's ARM repeat domain and the intrinsically disordered protein adenomatous polyposis coli (APC). APC is a giant multivalent scaffold that brings together the different components of the so-called "ß-catenin destruction complex", which drives ß-catenin degradation via the ubiquitin-proteasome pathway. Mutations and truncations in APC, resulting in loss of APC function and hence elevated ß-catenin levels and upregulation of Wnt signalling, are associated with numerous cancers including colorectal carcinomas. APC has a long intrinsically disordered region (IDR) that contains a series of 15-residue and 20-residue binding regions for ß-catenin. Here we explore the multivalent nature of the interaction of ß-catenin with the highest affinity APC repeat, both at equilibrium and under kinetic conditions. We use a combination of single-site substitutions, deletions and insertions to dissect the mechanism of molecular recognition and the roles of the three ß-catenin-binding subdomains of APC.

Structure-Based Discovery of Lipoteichoic Acid Synthase Inhibitors.

Lipoteichoic acid synthase (LtaS) is a key enzyme for the cell wall biosynthesis of Gram-positive bacteria. Gram-positive bacteria that lack lipoteichoic acid (LTA) exhibit impaired cell division and growth defects. Thus, LtaS appears to be an attractive antimicrobial target. The pharmacology around LtaS remains largely unexplored with only two small-molecule LtaS inhibitors reported, namely "compound 1771" and the Congo red dye. Structure-based drug discovery efforts against LtaS remain unattempted due to the lack of an inhibitor-bound structure of LtaS. To address this, we combined the use of a molecular docking technique with molecular dynamics (MD) simulations to model a plausible binding mode of compound 1771 to the extracellular catalytic domain of LtaS (eLtaS). The model was validated using alanine mutagenesis studies combined with isothermal titration calorimetry. Additionally, lead optimization driven by our computational model resulted in an improved version of compound 1771, namely, compound 4 which showed greater affinity for binding to eLtaS than compound 1771 in biophysical assays. Compound 4 reduced LTA production in S. aureus dose-dependently, induced aberrant morphology as seen for LTA-deficient bacteria, and significantly reduced bacteria titers in the lung of mice infected with S. aureus. Analysis of our MD simulation trajectories revealed the possible formation of a transient cryptic pocket in eLtaS. Virtual screening (VS) against the cryptic pocket led to the identification of a new class of inhibitors that could potentiate ß-lactams against methicillin-resistant S. aureus. Our overall workflow and data should encourage further drug design campaign against LtaS. Finally, our work reinforces the importance of considering protein conformational flexibility to a successful VS endeavor.

Unraveling the Mechanics of a Repeat-Protein Nanospring: From Folding of Individual Repeats to Fluctuations of the Superhelix.

Tandem-repeat proteins comprise small secondary structure motifs that stack to form one-dimensional arrays with distinctive mechanical properties that are proposed to direct their cellular functions. Here, we use single-molecule optical tweezers to study the folding of consensus-designed tetratricopeptide repeats (CTPRs), superhelical arrays of short helix-turn-helix motifs. We find that CTPRs display a spring-like mechanical response in which individual repeats undergo rapid equilibrium fluctuations between partially folded and unfolded conformations. We rationalize the force response using Ising models and dissect the folding pathway of CTPRs under mechanical load, revealing how the repeat arrays form from the center toward both termini simultaneously. Most strikingly, we also directly observe the protein's superhelical tertiary structure in the force signal. Using protein engineering, crystallography, and single-molecule experiments, we show that the superhelical geometry can be altered by carefully placed amino acid substitutions, and we examine how these sequence changes affect intrinsic repeat stability and inter-repeat coupling. Our findings provide the means to dissect and modulate repeat-protein stability and dynamics, which will be essential for researchers to understand the function of natural repeat proteins and to exploit artificial repeats proteins in nanotechnology and biomedical applications.

The Pathological G51D Mutation in Alpha-Synuclein Oligomers Confers Distinct Structural Attributes and Cellular Toxicity.

A wide variety of oligomeric structures are formed during the aggregation of proteins associated with neurodegenerative diseases. Such soluble oligomers are believed to be key toxic species in the related disorders; therefore, identification of the structural determinants of toxicity is of upmost importance. Here, we analysed toxic oligomers of α-synuclein and its pathological variants in order to identify structural features that could be related to toxicity and found a novel structural polymorphism within G51D oligomers. These G51D oligomers can adopt a variety of ß-sheet-rich structures with differing degrees of α-helical content, and the helical structural content of these oligomers correlates with the level of induced cellular dysfunction in SH-SY5Y cells. This structure-function relationship observed in α-synuclein oligomers thus presents the α-helical structure as another potential structural determinant that may be linked with cellular toxicity in amyloid-related proteins.

Accessible high-performance double nanohole tweezers.

Nanohole optical tweezers have been used by several groups to trap and analyze proteins. In this work, we demonstrate that it is possible to create high-performance double nanohole (DNH) substrates for trapping proteins without the need for any top-down approaches (such as electron microscopy or focused-ion beam milling). Using polarization analysis, we identify DNHs as well as determine their orientation and then use them for trapping. We are also able to identify other hole configurations, such as single, trimers and other clusters. We explore changing the substrate from glass to polyvinyl chloride to enhance trapping ability, showing 7 times lower minimum trapping power, which we believe is due to reduced surface repulsion. Finally, we present tape exfoliation as a means to expose DNHs without damaging sonication or chemical methods. Overall, these approaches make high quality optical trapping using DNH structures accessible to a broad scientific community.

Exploring the binding of rationally engineered tandem-repeat proteins to E3 ubiquitin ligase Keap1.

The process of displaying functional peptides by 'grafting' them onto loops of a stable protein scaffold can be used to impart binding affinity for a target, but it can be difficult to predict the affinity of the grafted peptide and the effect of grafting on scaffold stability. In this study, we show that a series of peptides that bind to the E3 ubiquitin ligase Keap1 can be grafted into the inter-repeat loop of a consensus-designed tetratricopeptide repeat (CTPR) protein resulting in proteins with high stability. We found that these CTPR-grafted peptides had similar affinities to their free peptide counterparts and achieved a low nanomolar range. This result is likely due to a good structural match between the inter-repeat loop of the CTPR and the Keap1-binding peptide. The grafting process led to the discovery of a new Keap1-binding peptide, Ac-LDPETGELL-NH2, with low nanomolar affinity for Keap1, highlighting the potential of the repeat-protein class for application in peptide display.

Parallel and Sequential Pathways of Molecular Recognition of a Tandem-Repeat Protein and Its Intrinsically Disordered Binding Partner.

The Wnt signalling pathway plays an important role in cell proliferation, differentiation, and fate decisions in embryonic development and the maintenance of adult tissues. The twelve armadillo (ARM) repeat-containing protein ß-catenin acts as the signal transducer in this pathway. Here, we investigated the interaction between ß-catenin and the intrinsically disordered transcription factor TCF7L2, comprising a very long nanomolar-affinity interface of approximately 4800 Å2 that spans ten of the twelve ARM repeats of ß-catenin. First, a fluorescence reporter system for the interaction was engineered and used to determine the kinetic rate constants for the association and dissociation. The association kinetics of TCF7L2 and ß-catenin were monophasic and rapid (7.3 ± 0.1 × 107 M-1·s-1), whereas dissociation was biphasic and slow (5.7 ± 0.4 × 10-4 s-1, 15.2 ± 2.8 × 10-4 s-1). This reporter system was then combined with site-directed mutagenesis to investigate the striking variability in the conformation adopted by TCF7L2 in the three different crystal structures of the TCF7L2-ß-catenin complex. We found that the mutation had very little effect on the association kinetics, indicating that most interactions form after the rate-limiting barrier for association. Mutations of the N- and C-terminal subdomains of TCF7L2 that adopt relatively fixed conformations in the crystal structures had large effects on the dissociation kinetics, whereas the mutation of the labile sub-domain connecting them had negligible effect. These results point to a two-site avidity mechanism of binding with the linker region forming a "fuzzy" complex involving transient contacts that are not site-specific. Strikingly, the two mutations in the N-terminal subdomain that had the largest effects on the dissociation kinetics showed two additional phases, indicating partial flux through an alternative dissociation pathway that is inaccessible to the wild type. The results presented here provide insights into the kinetics of the molecular recognition of a long intrinsically disordered region with an elongated repeat-protein surface, a process found to involve parallel routes with sequential steps in each.

Testing the length limit of loop grafting in a helical repeat protein.

Alpha-helical repeat proteins such as consensus-designed tetratricopeptide repeats (CTPRs) are exceptionally stable molecules that are able to tolerate destabilizing sequence alterations and are therefore becoming increasingly valued as a modular platform for biotechnology and biotherapeutic applications. A simple approach to functionalize the CTPR scaffold that we are pioneering is the insertion of short linear motifs (SLiMs) into the loops between adjacent repeats. Here, we test the limits of the scaffold by inserting 17 highly diverse amino acid sequences of up to 58 amino acids in length into a two-repeat protein and examine the impact on protein folding, stability and solubility. The sequences include three SLiMs that bind oncoproteins and eleven naturally occurring linker sequences all predicted to be intrinsically disordered but with conformational preferences ranging from compact globules to expanded coils. We show that the loop-grafted proteins retain the native CTPR structure and are thermally stable with melting temperatures above 60 â€‹°C, despite the longest loop sequence being almost the same size as the CTPR scaffold itself (68 amino acids). Although the main determinant of the effect of stability was found to be loop length and was relatively insensitive to amino acid composition, the relationship between protein solubility and the loop sequences was more complex, with the presence of negatively charged amino acids enhancing the solubility. Our findings will help us to fully realize the potential of the repeat-protein scaffold, allowing a rational design approach to create artificial modular proteins with customized functional capabilities.

Engineering mono- and multi-valent inhibitors on a modular scaffold.

Here we exploit the simple, ultra-stable, modular architecture of consensus-designed tetratricopeptide repeat proteins (CTPRs) to create a platform capable of displaying both single as well as multiple functions and with diverse programmable geometrical arrangements by grafting non-helical short linear binding motifs (SLiMs) onto the loops between adjacent repeats. As proof of concept, we built synthetic CTPRs to bind and inhibit the human tankyrase proteins (hTNKS), which play a key role in Wnt signaling and are upregulated in cancer. A series of mono-valent and multi-valent hTNKS binders was assembled. To fully exploit the modular scaffold and to further diversify the multi-valent geometry, we engineered the binding modules with two different formats, one monomeric and the other trimeric. We show that the designed proteins are stable, correctly folded and capable of binding to and inhibiting the cellular activity of hTNKS leading to downregulation of the Wnt pathway. Multivalency in both the CTPR protein arrays and the hTNKS target results in the formation of large macromolecular assemblies, which can be visualized both in vitro and in the cell. When delivered into the cell by nanoparticle encapsulation, the multivalent CTPR proteins displayed exceptional activity. They are able to inhibit Wnt signaling where small molecule inhibitors have failed to date. Our results point to the tremendous potential of the CTPR platform to exploit a range of SLiMs and assemble synthetic binding molecules with built-in multivalent capabilities and precise, pre-programmed geometries.

Probing the unfolded protein response in long-lived naked mole-rats.

The long-living naked mole-rat (NMR) shows negligible senescence and resistance to age-associated diseases. Recent evidence, based on protein-level assays, suggests that enhanced protein homeostasis machinery contributes to NMR stress-resistance and longevity. Here, we develop NMR-specific, transcriptional assays for measuring the unfolded protein response (UPR), a component of ER proteostasis. By varying doses and response times of pharmacological ER stressors applied to NMR kidney fibroblasts, we probe the NMR UPR in detail, demonstrating that NMR fibroblasts have a higher UPR activation threshold compared to mouse fibroblasts under mild ER-stress induction; whereas temporal analysis reveals that severe ER-stress induction results in no comparative differences. Probing NMR UPR activation with our robust assays may lead to insights into the proteostasis and ageing relationship.

Diarylethene moiety as an enthalpy-entropy switch: photoisomerizable stapled peptides for modulating p53/MDM2 interaction.

Analogs of the known inhibitor (peptide pDI) of the p53/MDM2 protein-protein interaction are reported, which are stapled by linkers bearing a photoisomerizable diarylethene moiety. The corresponding photoisomers possess significantly different affinities to the p53-interacting domain of the human MDM2. Apparent dissociation constants are in the picomolar-to-low nanomolar range for those isomers with diarylethene in the "open" configuration, but up to eight times larger for the corresponding "closed" isomers. Spectroscopic, structural, and computational studies showed that the stapling linkers of the peptides contribute to their binding. Calorimetry revealed that the binding of the "closed" isomers is mostly enthalpy-driven, whereas the "open" photoforms bind to the protein stronger due to their increased binding entropy. The results suggest that conformational dynamics of the protein-peptide complexes may explain the differences in the thermodynamic profiles of the binding.