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.

Clonal Evolution in Patients with Hormone Receptor Positive, HER-2 Negative Breast Cancer Treated with Chemotherapy or CDK4/6 Inhibitors.

INTRODUCTION: Somatic evolution of the cancer genome resulting in genetically different subclones is thought to be involved in the development of treatment resistance but might also offer new therapeutic opportunities in metastatic breast cancer. No data are available if clonal evolution differs in patients treated with chemotherapy (chemo) or CDK4/6 inhibitors given with endocrine treatment (CE treatment). METHODS: We performed a prospective analysis of circulating tumor DNA (ctDNA) by targeted next-generation sequencing in 46 patients before the beginning of a systemic first-line (n = 37) or second-line (n = 9) treatment. Ct DNA was analyzed again upon disease progression. RESULTS: New mutations in ctDNA of patients with progressive disease were detected in 1/11 patients who started chemo, in 4/9 patients treated with chemo followed by CE maintenance treatment, and in 9/26 patients receiving CE therapy. The number of acquired new mutations did not differ significantly between the three therapy cohorts (all p values >0.05). However, in patients classified as secondary resistant (n = 37), occurrence of new mutations significantly differed between patients who started chemo (0/9) compared to patients treated with chemo followed by CE (4/11; p = 0.041) and patients receiving CE therapy (8/19; p = 0.024), respectively. CONCLUSION: Clonal evolution might differ significantly between metastatic breast cancer patients with hormone receptor positive and HER-2 negative disease treated with chemo or CDK4/6 inhibitors. These results should be confirmed in larger patient cohorts.

Carbonate fluxes by coccolithophore species between NW Africa and the Caribbean: Implications for the biological carbon pump.

Coccolithophores are among the most important calcifying pelagic organisms. To assess how coccolithophore species with different coccolith-carbonate mass and distinct ecological resilience to ocean warming will influence the "rain ratio" and the "biological carbon pump", 1 yr of species-specific coccolith-carbonate export fluxes were quantified using sediment traps moored at four sites between NW Africa and the Caribbean (i.e., CB-20°N/21°W, at 1214 m; M1-12°N/23°W, at 1150 m; M2-14°N/37°W, at 1235 m; M4-12°N/49°W, at 1130 m). Highest coccolith-CaCO3 fluxes at the westernmost site M4, where the nutricline is deepest along the tropical North Atlantic, were dominated by deep-dwelling small-sized coccolith species Florisphaera profunda and Gladiolithus flabellatus. Total coccolith-CaCO3 fluxes of 371 mg m-2 yr-1 at M4 were followed by 165 mg m-2 yr-1 at the north-easternmost CB, 130 mg m-2 yr-1 at M1, and 114 mg m-2 yr-1 at M2 in between. Coccoliths accounted for nearly half of the total carbonate flux at M4 (45%), much higher compared to 23% at M2 and 15% at M1 and CB. At site M4, highest ratios of coccolith-CaCO3 to particulate organic carbon fluxes and weak correlations between the carbonate of deep-dwelling species and particulate organic carbon suggest that increasing productivity in the lower photic zone in response to ocean warming might enhance the rain ratio and reduce the coccolith-ballasting efficiency. The resulting weakened biological carbon pump could, however, be counterbalanced by increasing frequency of Saharan dust outbreaks across the tropical Atlantic, providing mineral ballast as well as nutrients to fuel fast-blooming and ballast-efficient coccolithophore species.

Elective Discontinuation of CDK4/6 Inhibitors in Patients with Metastatic Hormone Receptor-Positive, Her-2-Negative Breast Cancer: A Retrospective Single-Center Experience.

INTRODUCTION: Cyclin-dependent 4/6 kinase (CDK4/6) inhibitors given with endocrine therapy until disease progression are standard of care in the treatment of women with advanced HR-positive Her-2-negative breast cancer. No data are available if therapy can be safely de-escalated to endocrine monotherapy in patients with long-lasting disease control. METHODS: We performed a retrospective analysis on the clinical course of 22 patients at our center who received CDK4/6 inhibitors with aromatase inhibitors or fulvestrant. All patients had at least stable disease for >6 months and made a joint decision with their provider to electively discontinue CDK4/6 inhibitors. Best objective response (BOR) at treatment discontinuation, progression-free survival, and re-treatment characteristics were recorded. RESULTS: Of 138 patients who received CDK4/6 inhibitors as first- or second-line therapy at our center, 22 met the inclusion criteria. Median duration of CDK4/6 treatment was 18 months (range 6-45). BOR was complete response in 1, partial response in 8, and stable disease in 13 patients. After a median duration of endocrine monotherapy of 9.5 months (range 5-44 months), 6 of 22 patients had progressive disease (1 local relapse and 5 systemic progression). All patients with disease progression had at least stable disease to chemotherapy (N = 1) or re-treatment with CDK4/6 inhibitors (N = 4). CONCLUSION: Elective discontinuation of CDK4/6 inhibitors is feasible in patients with long-lasting disease stabilization. This strategy should be evaluated in prospective trials.

BASE GROWTH TEMPERATURE AND PHYLLOCHRON FOR KIKUYU GRASS (Cenchrus clandestinas; Poaceae) / Temperatura base de crecimiento y filocrono para el pasto kikuyo (Cenchrus clandestinus; Poacea)

ABSTRACT In order to estimate the base temperature (Bt) of growth through the appearance of leaves and calculate the phyllochron for kikuyu grass, three plots were established on three farms in the Provincia of Ubaté (Cundinamarca, Colombia) located at different altitudes (2560, 2640, 3143 m. a. s. l.). Measurements were made in four cycles in a period of eight months. The Bt was estimated by the least coefficient of variation method using a second order regression model and the model obtained was validated by the cross-validation method. The Bt values for the first, second, third and fourth leafwere 4.02, 3.68, 3.93, and 3.62 °C, respectively. For the appearance of the first leaf, the kikuyu required more thermal time (TT) (97.5 accumulated growing degree days (AGDD)) than for the second (74.2 AGDD), third (73.8 AGDD) and fourth leaf (76.0 AGDD) (p <0.05). There were no differences in TT among farms (p> 0.05). There was a tendency to a greater number of days required to reach each leaf stage in the farm located at higher altitude and with lower mean temperature. The validation showed an adequate adjustment (r2 = 0.94) and a substantial concordance (CCC = 0.97) between the observed values and the predicted values for the estimated TT with the Bt value obtained for each leaf stage. The results ofBt for kikuyu grass obtained, will allow to make more precise predictions about the phyllochron and generate growth models close to reality.

RESUMEN Con el fin de estimar la temperatura base (Tb) de crecimiento a través de la aparición de hojas y calcular el filocrono para el pasto kikuyo, se establecieron tres parcelas en tres fincas de la provincia de Ubaté (Cundinamarca, Colombia) ubicadas a diferente altitud (2560, 2640, 3143 m. s. n. m.). Se realizaron cuatro ciclos de mediciones en un tiempo total de ocho meses. La Tb se estimó por el método de mínimo coeficiente de variación utilizando un modelo de regresión de segundo grado y el modelo obtenido se validó por el método de validación cruzada. La Tb para la primera, segunda, tercera y cuarta hoja fue 4,02, 3,68, 3,93 y 3,62 °C, respectivamente. El kikuyo requirió mayor tiempo térmico (TT) para la aparición de la primera hoja (97,5 grados día de crecimiento acumulados (GDCA)) que para la segunda (74,2 GDCA), tercera (73,8 GDCA) y cuarta hoja (76,0 GDCA) (p<0,05). No hubo diferencias en el TT entre fincas (p > 0,05). Hubo tendencia a un mayor número de días calendario requeridos para alcanzar cada estado de hoja en la finca ubicada a mayor altitud y con menor temperatura media. La validación mostró un adecuado ajuste (r2 = 0,94) y una concordancia sustancial (CCC= 0,97) entre los valores observados y los valores predichos para el TT estimado con los valores de Tb obtenidos para cada estado de hoja. Los resultados de Tb de crecimiento del pasto kikuyo obtenidos permiten realizar predicciones más precisas sobre el filocrono y generar modelos de crecimiento cercanos a la realidad.

HUWE1 employs a giant substrate-binding ring to feed and regulate its HECT E3 domain.

HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin-proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of Nematocida HUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates. The protein adopts a remarkable snake-like structure, where the C-terminal HECT domain heads an extended alpha-solenoid body that coils in on itself and houses various protein-protein interaction modules. Our integrative structural analysis shows that this ring structure is highly dynamic, enabling the flexible HECT domain to reach protein targets presented by the various acceptor sites. Together, our data demonstrate how HUWE1 is regulated by its unique structure, adapting a promiscuous E3 ligase to selectively target unassembled orphan proteins.

Review on the ecophysiology of important Andean fruits: Solanaceae / Revisión de la ecofisiología de frutos andinos importantes: Solanaceae

ABSTRACT The high Andean areas present ecophysiological conditions suitable for the cultivation of many fruit species, especially of the Solanaceae family. The objective of this review is to present important ecophysiological information on four fruit trees grown in cold climates: Cape gooseberry (Physalis peruviana L.), tree tomato (Solanum betaceum Cav.), lulo (Solanum quitoense Lam.), and sweet cucumber o pear melon (Solanum muricatum Aiton). The cape gooseberry is a species well adapted to cold tropical climate, it is grown between 1,800 and 2,700m a.s.l., with temperatures of 13 to 16°C. It is highly adapted to high solar radiation and to the abrupt changes between the day and night temperatures. It requires a precipitation of 1,000 to 1,800mm year-1 uniformly distributed throughout the year, and is sensitive to water deficit but also to waterlogging and strong winds. The tree tomato, in Colombia, produces better from 1,800 to 2,600m a.s.l., with temperatures between 13 and 20°C, annual rainfall between 1,500 and 2,000mm, relative humidity around 80%, and solar brightness of 1,800 to 2,300 hours/year; it does not resist strong winds, water deficit or waterlogging. The lulo requires environments with high precipitation (1,000 to 2,800mm) and penumbra because it loses a lot of water through transpiration but waterlogging also affects it; it grows well in areas between 1,600 to 2,400m a.s.l. and 16 to 24°C, with photosynthesis rates up to of 34.03µmol CO2 m-2 s-1. The sweet cucumber is of growing interest in many exotic fruit markets, it grows at 900-2,800m a.s.l. with temperatures <25°C and responds well to air enrichment with CO2.

RESUMEN Las zonas altoandinas presentan condiciones ecofisiológicas aptas para el cultivo de muchas especies frutales, especialmente, de la familia Solanaceae. El objetivo de este artículo de revisión de literatura fue reunir la información ecofisiológica importante sobre cuatro frutales, cultivados en clima frío: uchuva (Physalis peruviana L.), tomate de árbol (Solanum betaceum Cav.), lulo (Solanum quitoense Lam.) y pepino dulce (Solanum muricatum Aiton). La uchuva es una especie bien adaptada a clima frio, se cultiva entre los 1.800 y 2.700m s.n.m., con temperaturas de 13 a 16°C. Es altamente adaptada a una elevada radiación solar y al cambio brusco entre la temperatura del día y de la noche. Requiere una precipitación de 1.000 a 1.800mm año-1, distribuido uniformemente durante el año; es sensible al déficit hídrico, pero también al encharcamiento y a los fuertes vientos. El tomate de árbol, en Colombia, produce mejor de 1.800 a 2.600m s.n.m., con temperaturas entre 13 y 20°C, con una precipitación anual entre 1.500 y 2.000mm y humedad relativa alrededor del 80%, con un brillo solar de 1.800 a 2.300 horas/año; no resiste vientos fuertes, déficit hídrico, ni anegamiento. El lulo requiere ambientes con alta precipitación (1.000 a 2.800mm) y penumbra, porque pierde mucha agua por transpiración, pero el anegamiento también lo afecta; crece bien en zonas entre 1.600 a 2.400m s.n.m. y 16 a 24°C, con tasas de fotosíntesis hasta de 34,03µmol CO2 m-2 s-1. El pepino dulce está generando un creciente interés en muchos mercados de los frutos exóticos, crece en 900-2.800m s.n.m., con temperaturas <25°C y responde bien al enriquecimiento con CO2.

A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death.

BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.

Drugging all RAS isoforms with one pocket.

Activating mutations in the three human RAS genes, KRAS, NRAS and HRAS, are among the most common oncogenic drivers in human cancers. Covalent KRASG12C inhibitors, which bind to the switch II pocket in the 'off state' of KRAS, represent the first direct KRAS drugs that entered human clinical trials. However, the remaining 85% of non-KRASG12C-driven cancers remain undrugged as do NRAS and HRAS and no drugs targeting the 'on state' have been discovered so far. The switch I/II pocket is a second pocket for which the nanomolar inhibitor BI-2852 has been discovered. Here, we elucidate inhibitor binding modes in KRAS, NRAS and HRAS on and off and discuss future strategies to drug all RAS isoforms with this one pocket.

Targeting of Fumarate Hydratase from Mycobacterium tuberculosis Using Allosteric Inhibitors with a Dimeric-Binding Mode.

With the growing worldwide prevalence of antibiotic-resistant strains of tuberculosis (TB), new targets are urgently required for the development of treatments with novel modes of action. Fumarate hydratase (fumarase), a vulnerable component of the citric acid cycle in Mycobacterium tuberculosis (Mtb), is a metabolic target that could satisfy this unmet demand. A key challenge in the targeting of Mtb fumarase is its similarity to the human homolog, which shares an identical active site. A potential solution to this selectivity problem was previously found in a high-throughput screening hit that binds in a nonconserved allosteric site. In this work, a structure-activity relationship study was carried out with the determination of further structural biology on the lead series, affording derivatives with sub-micromolar inhibition. Further, the screening of this series against Mtb in vitro identified compounds with potent minimum inhibitory concentrations.

KRAS Binders Hidden in Nature.

Natural products have proven to be a rich source of molecular architectures for drugs. Here, an integrated approach to natural product screening is proposed, which uncovered eight new natural product scaffolds for KRAS-the most frequently mutated oncogenic driver in human cancers, which has remained thus far undrugged. The approach combines aspects of virtual screening, fragment-based screening, structure-activity relationships (SAR) by NMR, and structure-based drug discovery to overcome the limitations in traditional natural product approaches. By using our approach, a new "snugness of fit" scoring function and the first crystal-soaking system of the active form of KRASG12D , the protein-ligand X-ray structures of a tricyclic indolopyrrole fungal alkaloid and an indoloisoquinolinone have been successfully elucidated. The natural product KRAS hits discovered provide fruitful ground for the optimization of highly potent natural-product-based inhibitors of the active form of oncogenic RAS. This integrated approach for screening natural products also holds promise for other "undruggable" targets.

Balancing Specificity and Promiscuity in Enzyme Evolution: Multidimensional Activity Transitions in the Alkaline Phosphatase Superfamily.

Highly proficient, promiscuous enzymes can be springboards for functional evolution, able to avoid loss of function during adaptation by their capacity to promote multiple reactions. We employ a systematic comparative study of structure, sequence, and substrate specificity to track the evolution of specificity and reactivity between promiscuous members of clades of the alkaline phosphatase (AP) superfamily. Construction of a phylogenetic tree of protein sequences maps out the likely transition zone between arylsulfatases (ASs) and phosphonate monoester hydrolases (PMHs). Kinetic analysis shows that all enzymes characterized have four chemically distinct phospho- and sulfoesterase activities, with rate accelerations ranging from 1011- to 1017-fold for their primary and 109- to 1012-fold for their promiscuous reactions, suggesting that catalytic promiscuity is widespread in the AP-superfamily. This functional characterization and crystallography reveal a novel class of ASs that is so similar in sequence to known PMHs that it had not been recognized as having diverged in function. Based on analysis of snapshots of catalytic promiscuity "in transition", we develop possible models that would allow functional evolution and determine scenarios for trade-off between multiple activities. For the new ASs, we observe largely invariant substrate specificity that would facilitate the transition from ASs to PMHs via trade-off-free molecular exaptation, that is, evolution without initial loss of primary activity and specificity toward the original substrate. This ability to bypass low activity generalists provides a molecular solution to avoid adaptive conflict.

Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset.

The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and diester hydrolyses were only marginally affected (≤50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E•S, enzyme-substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate (ßleavinggroup from -1.08 to -0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes.

Structure of the human myostatin precursor and determinants of growth factor latency.

Myostatin, a key regulator of muscle mass in vertebrates, is biosynthesised as a latent precursor in muscle and is activated by sequential proteolysis of the pro-domain. To investigate the molecular mechanism by which pro-myostatin remains latent, we have determined the structure of unprocessed pro-myostatin and analysed the properties of the protein in its different forms. Crystal structures and SAXS analyses show that pro-myostatin adopts an open, V-shaped structure with a domain-swapped arrangement. The pro-mature complex, after cleavage of the furin site, has significantly reduced activity compared with the mature growth factor and persists as a stable complex that is resistant to the natural antagonist follistatin. The latency appears to be conferred by a number of distinct features that collectively stabilise the interaction of the pro-domains with the mature growth factor, enabling a regulated stepwise activation process, distinct from the prototypical pro-TGF-ß1. These results provide a basis for understanding the effect of missense mutations in pro-myostatin and pave the way for the design of novel myostatin inhibitors.

Author Correction: Temperature dependence of protein-water interactions in a gated yeast aquaporin.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Temperature dependence of protein-water interactions in a gated yeast aquaporin.

Regulation of aquaporins is a key process of living organisms to counteract sudden osmotic changes. Aqy1, which is a water transporting aquaporin of the yeast Pichia pastoris, is suggested to be gated by chemo-mechanical stimuli as a protective regulatory-response against rapid freezing. Here, we tested the influence of temperature by determining the X-ray structure of Aqy1 at room temperature (RT) at 1.3 Å resolution, and by exploring the structural dynamics of Aqy1 during freezing through molecular dynamics simulations. At ambient temperature and in a lipid bilayer, Aqy1 adopts a closed conformation that is globally better described by the RT than by the low-temperature (LT) crystal structure. Locally, for the blocking-residue Tyr31 and the water molecules inside the pore, both LT and RT data sets are consistent with the positions observed in the simulations at room-temperature. Moreover, as the temperature was lowered, Tyr31 adopted a conformation that more effectively blocked the channel, and its motion was accompanied by a temperature-driven rearrangement of the water molecules inside the channel. We therefore speculate that temperature drives Aqy1 from a loosely- to a tightly-blocked state. This analysis provides high-resolution structural evidence of the influence of temperature on membrane-transport channels.

Structural analyses of von Willebrand factor C domains of collagen 2A and CCN3 reveal an alternative mode of binding to bone morphogenetic protein-2.

Bone morphogenetic proteins (BMPs) are secreted growth factors that promote differentiation processes in embryogenesis and tissue development. Regulation of BMP signaling involves binding to a variety of extracellular proteins, among which are many von Willebrand factor C (vWC) domain-containing proteins. Although the crystal structure of the complex of crossveinless-2 (CV-2) vWC1 and BMP-2 previously revealed one mode of the vWC/BMP-binding mechanism, other vWC domains may bind to BMP differently. Here, using X-ray crystallography, we present for the first time structures of the vWC domains of two proteins thought to interact with BMP-2: collagen IIA and matricellular protein CCN3. We found that these two vWC domains share a similar N-terminal fold that differs greatly from that in CV-2 vWC, which comprises its BMP-2-binding site. We analyzed the ability of these vWC domains to directly bind to BMP-2 and detected an interaction only between the collagen IIa vWC and BMP-2. Guided by the collagen IIa vWC domain crystal structure and conservation of surface residues among orthologous domains, we mapped the BMP-binding epitope on the subdomain 1 of the vWC domain. This binding site is different from that previously observed in the complex between CV-2 vWC and BMP-2, revealing an alternative mode of interaction between vWC domains and BMPs.

Macrocyclized Extended Peptides: Inhibiting the Substrate-Recognition Domain of Tankyrase.

We report a double-click macrocyclization approach for the design of constrained peptide inhibitors having non-helical or extended conformations. Our targets are the tankyrase proteins (TNKS), poly(ADP-ribose) polymerases (PARP) that regulate Wnt signaling by targeting Axin for degradation. TNKS are deregulated in many different cancer types, and inhibition of TNKS therefore represents an attractive therapeutic strategy. However, clinical development of TNKS-specific PARP catalytic inhibitors is challenging due to off-target effects and cellular toxicity. We instead targeted the substrate-recognition domain of TNKS, as it is unique among PARP family members. We employed a two-component strategy, allowing peptide and linker to be separately engineered and then assembled in a combinatorial fashion via click chemistry. Using the consensus substrate-peptide sequence as a starting point, we optimized the length and rigidity of the linker and its position along the peptide. Optimization was further guided by high-resolution crystal structures of two of the macrocyclized peptides in complex with TNKS. This approach led to macrocyclized peptides with submicromolar affinities for TNKS and high proteolytic stability that are able to disrupt the interaction between TNKS and Axin substrate and to inhibit Wnt signaling in a dose-dependent manner. The peptides therefore represent a promising starting point for a new class of substrate-competitive inhibitors of TNKS with potential for suppressing Wnt signaling in cancer. Moreover, by demonstrating the application of the double-click macrocyclization approach to non-helical, extended, or irregularly structured peptides, we greatly extend its potential and scope, especially given the frequency with which such motifs mediate protein-protein interactions.

Engineering Archeal Surrogate Systems for the Development of Protein-Protein Interaction Inhibitors against Human RAD51.

Protein-protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based drug discovery approaches to tackle PPIs are often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering to "humanise" thermophilic archeal surrogate proteins as targets for small-molecule inhibitor discovery and to exemplify this approach in the development of inhibitors against the PPI between the recombinase RAD51 and tumour suppressor BRCA2. As human RAD51 has proved impossible to produce in a form that is compatible with the requirements of fragment-based drug discovery, we have developed a surrogate protein system using RadA from Pyrococcus furiosus. Using a monomerised RadA as our starting point, we have adopted two parallel and mutually instructive approaches to mimic the human enzyme: firstly by mutating RadA to increase sequence identity with RAD51 in the BRC repeat binding sites, and secondly by generating a chimeric archaeal human protein. Both approaches generate proteins that interact with a fourth BRC repeat with affinity and stoichiometry comparable to human RAD51. Stepwise humanisation has also allowed us to elucidate the determinants of RAD51 binding to BRC repeats and the contributions of key interacting residues to this interaction. These surrogate proteins have enabled the development of biochemical and biophysical assays in our ongoing fragment-based small-molecule inhibitor programme and they have allowed us to determine hundreds of liganded structures in support of our structure-guided design process, demonstrating the feasibility and advantages of using archeal surrogates to overcome difficulties in handling human proteins.

Structure and activation of pro-activin A.

Activins are growth factors with multiple roles in the development and homeostasis. Like all TGF-ß family of growth factors, activins are synthesized as large precursors from which mature dimeric growth factors are released proteolytically. Here we have studied the activation of activin A and determined crystal structures of the unprocessed precursor and of the cleaved pro-mature complex. Replacing the natural furin cleavage site with a HRV 3C protease site, we show how the protein gains its bioactivity after proteolysis and is as active as the isolated mature domain. The complex remains associated in conditions used for biochemical analysis with a dissociation constant of 5 nM, but the pro-domain can be actively displaced from the complex by follistatin. Our high-resolution structures of pro-activin A share features seen in the pro-TGF-ß1 and pro-BMP-9 structures, but reveal a new oligomeric arrangement, with a domain-swapped, cross-armed conformation for the protomers in the dimeric protein.