Modular binder technology by NGS-aided, high-resolution selection in yeast of designed armadillo modules.

Establishing modular binders as diagnostic detection agents represents a cost- and time-efficient alternative to the commonly used binders that are generated one molecule at a time. In contrast to these conventional approaches, a modular binder can be designed in silico from individual modules to, in principle, recognize any desired linear epitope without going through a selection and hit-validation process, given a set of preexisting, amino acid-specific modules. Designed armadillo repeat proteins (dArmRP) have been developed as modular binder scaffolds, and we report here the generation of highly specific dArmRP modules by yeast surface display selection, performed on a rationally designed dArmRP library. A selection strategy was developed to distinguish the binding difference resulting from a single amino acid mutation in the target peptide. Our reverse-competitor strategy introduced here employs the designated target as a competitor to increase the sensitivity when separating specific from cross-reactive binders that show similar affinities for the target peptide. With this switch in selection focus from affinity to specificity, we found that the enrichment during this specificity sort is indicative of the desired phenotype, regardless of the binder abundance. Hence, deep sequencing of the selection pools allows retrieval of phenotypic hits with only 0.1% abundance in the selectivity sort pool from the next-generation sequencing data alone. In a proof-of-principle study, a binder was created by replacing all corresponding wild-type modules with a newly selected module, yielding a binder with very high affinity for the designated target that has been successfully validated as a detection agent in western blot analysis.

Investigation into the difference in mitochondrial-cytosolic calcium coupling between adult cardiomyocyte and hiPSC-CM using a novel multifunctional genetic probe.

Ca2+ cycling plays a critical role in regulating cardiomyocyte (CM) function under both physiological and pathological conditions. Mitochondria have been implicated in Ca2+ handling in adult cardiomyocytes (ACMs). However, little is known about their role in the regulation of Ca2+ dynamics in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). In the present study, we developed a multifunctional genetically encoded Ca2+ probe capable of simultaneously measuring cytosolic and mitochondrial Ca2+ in real time. Using this novel probe, we determined and compared mitochondrial Ca2+ activity and the coupling with cytosolic Ca2+ dynamics in hiPSC-CMs and ACMs. Our data showed that while ACMs displayed a highly coordinated beat-by-beat response in mitochondrial Ca2+ in sync with cytosolic Ca2+, hiPSC-CMs showed high cell-wide variability in mitochondrial Ca2+ activity that is poorly coordinated with cytosolic Ca2+. We then revealed that mitochondrial-sarcoplasmic reticulum (SR) tethering, as well as the inter-mitochondrial network connection, is underdeveloped in hiPSC-CM compared to ACM, which may underlie the observed spatiotemporal decoupling between cytosolic and mitochondrial Ca2+ dynamics. Finally, we showed that knockdown of mitofusin-2 (Mfn2), a protein tethering mitochondria and SR, led to reduced cytosolic-mitochondrial Ca2+ coupling in ACMs, albeit to a lesser degree compared to hiPSC-CMs, suggesting that Mfn2 is a potential engineering target for improving mitochondrial-cytosolic Ca2+ coupling in hiPSC-CMs. Physiological relevance: The present study will advance our understanding of the role of mitochondria in Ca2+ handling and cycling in CMs, and guide the development of hiPSC-CMs for healing injured hearts.

MitoQ regulates redox-related noncoding RNAs to preserve mitochondrial network integrity in pressure-overload heart failure.

Evidence suggests that mitochondrial network integrity is impaired in cardiomyocytes from failing hearts. While oxidative stress has been implicated in heart failure (HF)-associated mitochondrial remodeling, the effect of mitochondrial-targeted antioxidants, such as mitoquinone (MitoQ), on the mitochondrial network in a model of HF (e.g., pressure overload) has not been demonstrated. Furthermore, the mechanism of this regulation is not completely understood with an emerging role for posttranscriptional regulation via long noncoding RNAs (lncRNAs). We hypothesized that MitoQ preserves mitochondrial fusion proteins (i.e., mitofusin), likely through redox-sensitive lncRNAs, leading to improved mitochondrial network integrity in failing hearts. To test this hypothesis, 8-wk-old C57BL/6J mice were subjected to ascending aortic constriction (AAC), which caused substantial left ventricular (LV) chamber remodeling and remarkable contractile dysfunction in 1 wk. Transmission electron microscopy and immunostaining revealed defective intermitochondrial and mitochondrial-sarcoplasmic reticulum ultrastructure in AAC mice compared with sham-operated animals, which was accompanied by elevated oxidative stress and suppressed mitofusin (i.e., Mfn1 and Mfn2) expression. MitoQ (1.36 mg·day-1·mouse-1, 7 consecutive days) significantly ameliorated LV dysfunction, attenuated Mfn2 downregulation, improved interorganellar contact, and increased metabolism-related gene expression. Moreover, our data revealed that MitoQ alleviated the dysregulation of an Mfn2-associated lncRNA (i.e., Plscr4). In summary, the present study supports a unique mechanism by which MitoQ improves myocardial intermitochondrial and mitochondrial-sarcoplasmic reticulum (SR) ultrastructural remodeling in HF by maintaining Mfn2 expression via regulation by an lncRNA. These findings underscore the important role of lncRNAs in the pathogenesis of HF and the potential of targeting them for effective HF treatment.NEW & NOTEWORTHY We have shown that MitoQ improves cardiac mitochondrial network integrity and mitochondrial-SR alignment in a pressure-overload mouse heart-failure model. This may be occurring partly through preventing the dysregulation of a redox-sensitive lncRNA-microRNA pair (i.e., Plscr4-miR-214) that results in an increase in mitofusin-2 expression.

Precisely Control Mitochondria with Light to Manipulate Cell Fate Decision.

Mitochondrial dysfunction has been implicated in many pathological conditions and diseases. The normal functioning of mitochondria relies on maintaining the inner mitochondrial membrane potential (also known as ΔΨm) that is essential for ATP synthesis, Ca2+ homeostasis, redox balance, and regulation of other key signaling pathways such as mitophagy and apoptosis. However, the detailed mechanisms by which ΔΨm regulates cellular function remain incompletely understood, partially because of the difficulty of manipulating ΔΨm with spatiotemporal resolution, reversibility, or cell type specificity. To address this need, we have developed a next generation optogenetic-based technique for controllable mitochondrial depolarization with light. We demonstrate successful targeting of the heterologous channelrhodopsin-2 fusion protein to the inner mitochondrial membrane and formation of functional cationic channels capable of light-induced selective ΔΨm depolarization and mitochondrial autophagy. Importantly, we for the first time, to our knowledge, show that optogenetic-mediated mitochondrial depolarization can be well controlled to differentially influence the fate of cells expressing mitochondrial channelrhodopsin-2; whereas sustained moderate light illumination induces substantial apoptotic cell death, transient mild light illumination elicits cytoprotection via mitochondrial preconditioning. Finally, we show that Parkin overexpression exacerbates, instead of ameliorating, mitochondrial depolarization-mediated cell death in HeLa cells. In summary, we provide evidence that the described mitochondrial-targeted optogenetics may have a broad application for studying the role of mitochondria in regulating cell function and fate decision.

Curvature of designed armadillo repeat proteins allows modular peptide binding.

Designed armadillo repeat proteins (dArmRPs) were developed to create a modular peptide binding technology where each of the structural repeats binds two residues of the target peptide. An essential prerequisite for such a technology is a dArmRP geometry that matches the peptide bond length. To this end, we determined a large set (n=27) of dArmRP X-ray structures, of which 12 were previously unpublished, to calculate curvature parameters that define their geometry. Our analysis shows that consensus dArmRPs exhibit curvatures close to the optimal range for modular peptide recognition. Binding of peptide ligands can induce a curvature within the desired range, as confirmed by single-molecule FRET experiments in solution. On the other hand, computationally designed ArmRPs, where side chains have been chosen with the intention to optimally fit into a geometrically optimized backbone, turned out to be more divergent in reality, and thus not suitable for continuous peptide binding. Furthermore, we show that the formation of a crystal lattice can induce small but significant deviations from the curvature adopted in solution, which can interfere with the evaluation of repeat protein scaffolds when high accuracy is required. This study corroborates the suitability of consensus dArmRPs as a scaffold for the development of modular peptide binders.

Advances in the design and engineering of peptide-binding repeat proteins.

The specific recognition of peptides, which we define to include unstructured regions or denatured forms of proteins, is an intrinsic part of a multitude of biochemical assays and procedures. Many cellular interactions are also based on this principle as well. While it would be highly desirable to have a stockpile of sequence-specific binders for essentially any sequence, a de novo selection of individual binders against every possible target peptide sequence would be rather difficult to reduce to practice. Modular peptide binders could overcome this problem, as preselected and/or predesigned modules could be reused for the generation of new binders and thereby revolutionize the generation of binding proteins. This minireview summarizes advances in the development of peptide binders and possible scaffolds for their design.

KnowLife: a versatile approach for constructing a large knowledge graph for biomedical sciences.

BACKGROUND: Biomedical knowledge bases (KB's) have become important assets in life sciences. Prior work on KB construction has three major limitations. First, most biomedical KBs are manually built and curated, and cannot keep up with the rate at which new findings are published. Second, for automatic information extraction (IE), the text genre of choice has been scientific publications, neglecting sources like health portals and online communities. Third, most prior work on IE has focused on the molecular level or chemogenomics only, like protein-protein interactions or gene-drug relationships, or solely address highly specific topics such as drug effects. RESULTS: We address these three limitations by a versatile and scalable approach to automatic KB construction. Using a small number of seed facts for distant supervision of pattern-based extraction, we harvest a huge number of facts in an automated manner without requiring any explicit training. We extend previous techniques for pattern-based IE with confidence statistics, and we combine this recall-oriented stage with logical reasoning for consistency constraint checking to achieve high precision. To our knowledge, this is the first method that uses consistency checking for biomedical relations. Our approach can be easily extended to incorporate additional relations and constraints. We ran extensive experiments not only for scientific publications, but also for encyclopedic health portals and online communities, creating different KB's based on different configurations. We assess the size and quality of each KB, in terms of number of facts and precision. The best configured KB, KnowLife, contains more than 500,000 facts at a precision of 93% for 13 relations covering genes, organs, diseases, symptoms, treatments, as well as environmental and lifestyle risk factors. CONCLUSION: KnowLife is a large knowledge base for health and life sciences, automatically constructed from different Web sources. As a unique feature, KnowLife is harvested from different text genres such as scientific publications, health portals, and online communities. Thus, it has the potential to serve as one-stop portal for a wide range of relations and use cases. To showcase the breadth and usefulness, we make the KnowLife KB accessible through the health portal (http://knowlife.mpi-inf.mpg.de).

An Innovative Mitochondrial-targeted Gene Therapy for Cancer Treatment.

Targeting cancer cell mitochondria holds great therapeutic promise, yet current strategies to specifically and effectively destroy cancer mitochondria in vivo are limited. Here, we introduce mLumiOpto, an innovative mitochondrial-targeted luminoptogenetics gene therapy designed to directly disrupt the inner mitochondrial membrane (IMM) potential and induce cancer cell death. We synthesize a blue light-gated channelrhodopsin (CoChR) in the IMM and co-express a blue bioluminescence-emitting Nanoluciferase (NLuc) in the cytosol of the same cells. The mLumiOpto genes are selectively delivered to cancer cells in vivo by using adeno-associated virus (AAV) carrying a cancer-specific promoter or cancer-targeted monoclonal antibody-tagged exosome-associated AAV. Induction with NLuc luciferin elicits robust endogenous bioluminescence, which activates mitochondrial CoChR, triggering cancer cell IMM permeability disruption, mitochondrial damage, and subsequent cell death. Importantly, mLumiOpto demonstrates remarkable efficacy in reducing tumor burden and killing tumor cells in glioblastoma or triple-negative breast cancer xenografted mouse models. These findings establish mLumiOpto as a novel and promising therapeutic strategy by targeting cancer cell mitochondria in vivo.

mLumiOpto is a Mitochondrial-Targeted Gene Therapy for Treating Cancer.

Mitochondria are important in various aspects of cancer development and progression. Targeting mitochondria in cancer cells holds great therapeutic promise, yet current strategies to specifically and effectively destroy cancer mitochondria in vivo are limited. Here, we developed mLumiOpto, an innovative mitochondrial-targeted luminoptogenetics gene therapy designed to directly disrupt the inner mitochondrial membrane (IMM) potential and induce cancer cell death. The therapeutic approach included synthesis of a blue light-gated cationic channelrhodopsin (CoChR) in the IMM and co-expression of a blue bioluminescence-emitting nanoluciferase (NLuc) in the cytosol of the same cells. The mLumiOpto genes were selectively delivered to cancer cells in vivo by an adeno-associated virus (AAV) carrying a cancer-specific promoter or cancer-targeted monoclonal antibody-tagged exosome-associated AAV (mAb-Exo-AAV). Induction with NLuc luciferin elicited robust endogenous bioluminescence, which activated CoChR, triggering cancer cell mitochondrial depolarization and subsequent cell death. Importantly, mLumiOpto demonstrated remarkable efficacy in reducing tumor burden and killing tumor cells in glioblastoma and triple-negative breast cancer xenograft mouse models. Furthermore, the approach induced an anti-tumor immune response, increasing infiltration of dendritic cells and CD8+ T cells in the tumor microenvironment. These findings establish mLumiOpto as a promising therapeutic strategy by targeting cancer cell mitochondria in vivo.

Characterization of the far-red fluorescent probe MitoView 633 for dynamic mitochondrial membrane potential measurement.

Introduction: MitoView 633, a far-red fluorescent dye, exhibits the ability to accumulate within mitochondria in a membrane potential-dependent manner, as described by the Nernst equation. This characteristic renders it a promising candidate for bioenergetics studies, particularly as a robust indicator of mitochondrial membrane potential (DYm). Despite its great potential, its utility in live cell imaging has not been well characterized. Methods: This study seeks to characterize the spectral properties of MitoView 633 in live cells and evaluate its mitochondrial staining, resistance to photobleaching, and dynamics during DYm depolarization. The co-staining and imaging of MitoView 633 with other fluorophores such as MitoSOX Red and Fluo-4 AM were also examined in cardiomyocytes using confocal microscopy. Results and Discussion: Spectrum analysis showed that MitoView 633 emission could be detected at 660 ± 50 nm, and exhibited superior thermal stability compared to tetramethylrhodamine methyl ester (TMRM), a commonly used DYm indicator, which emits at 605 ± 25 nm. Confocal imaging unequivocally illustrated MitoView 633's specific localization within the mitochondrial matrix, corroborated by its colocalization with MitoTracker Green, a well-established mitochondrial marker. Furthermore, our investigation revealed that MitoView 633 exhibited minimal photobleaching at the recommended in vitro concentrations. Additionally, the dynamics of MitoView 633 fluoresce during carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, a mitochondrial uncoupler)-induced DYm depolarization mirrored that of TMRM. Importantly, MitoView 633 demonstrated compatibility with co-staining alongside MitoSOX Red and Fluo-4 AM, enabling concurrent monitoring of DYm, mitochondrial ROS, and cytosolic Ca2+ in intact cells. Conclusion: These findings collectively underscore MitoView 633 as a superb molecular probe for the singular or combined assessment of DYm and other indicators in live cell imaging applications.

Unexpected dynamics in femtomolar complexes of binding proteins with peptides.

Ultra-tight binding is usually observed for proteins associating with rigidified molecules. Previously, we demonstrated that femtomolar binders derived from the Armadillo repeat proteins (ArmRPs) can be designed to interact very tightly with fully flexible peptides. Here we show for ArmRPs with four and seven sequence-identical internal repeats that the peptide-ArmRP complexes display conformational dynamics. These dynamics stem from transient breakages of individual protein-residue contacts that are unrelated to overall unbinding. The labile contacts involve electrostatic interactions. We speculate that these dynamics allow attaining very high binding affinities, since they reduce entropic losses. Importantly, only NMR techniques can pick up these local events by directly detecting conformational exchange processes without complications from changes in solvent entropy. Furthermore, we demonstrate that the interaction surface of the repeat protein regularizes upon peptide binding to become more compatible with the peptide geometry. These results provide novel design principles for ultra-tight binders.

Optogenetic Studies of Mitochondria.

While optogenetic approaches have been widely used for remote control of cell membrane excitability and intracellular signaling pathways, their application in mitochondrial study has been limited, largely due to the challenge of effectively and specifically expressing heterologous light-gated rhodopsin channels in the mitochondria. Here, we describe the methods for expressing functional channelrhodopsin 2 (ChR2) proteins in the mitochondrial inner membrane with an unusually long mitochondrial leading sequence and characterizing optogenetic-mediated mitochondrial membrane potential (ΔΨm) depolarization. We then illustrate how this next-generation optogenetic approach can be used to study the effect of ΔΨm on mitochondrial functions such as mitophagy, programed cell death, and preconditioning-mediated cytoprotection. We anticipate that this innovative technology will enable new insights into the mechanisms by which changes in ΔΨm differentially impacts mitochondrial and cellular functions.

Cardiac calcium regulation in human induced pluripotent stem cell cardiomyocytes: Implications for disease modeling and maturation.

Human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) are based on ground-breaking technology that has significantly impacted cardiovascular research. They provide a renewable source of human cardiomyocytes for a variety of applications including in vitro disease modeling and drug toxicity testing. Cardiac calcium regulation plays a critical role in the cardiomyocyte and is often dysregulated in cardiovascular disease. Due to the limited availability of human cardiac tissue, calcium handling and its regulation have most commonly been studied in the context of animal models. hiPSC-CMs can provide unique insights into human physiology and pathophysiology, although a remaining limitation is the relative immaturity of these cells compared to adult cardiomyocytes Therefore, this field is rapidly developing techniques to improve the maturity of hiPSC-CMs, further establishing their place in cardiovascular research. This review briefly covers the basics of cardiomyocyte calcium cycling and hiPSC technology, and will provide a detailed description of our current understanding of calcium in hiPSC-CMs.

Antibody-Drug Conjugate to Treat Meningiomas.

Meningiomas are primary tumors of the central nervous system with high recurrence. It has been reported that somatostatin receptor 2 (SSTR2) is highly expressed in most meningiomas, but there is no effective targeted therapy approved to control meningiomas. This study aimed to develop and evaluate an anti-SSTR2 antibody-drug conjugate (ADC) to target and treat meningiomas. The meningioma targeting, circulation stability, toxicity, and anti-tumor efficacy of SSTR2 ADC were evaluated using cell lines and/or an intracranial xenograft mouse model. The flow cytometry analysis showed that the anti-SSTR2 mAb had a high binding rate of >98% to meningioma CH157-MN cells but a low binding rate of <5% to the normal arachnoidal AC07 cells. The In Vivo Imaging System (IVIS) imaging demonstrated that the Cy5.5-labeled ADC targeted and accumulated in meningioma xenograft but not in normal organs. The pharmacokinetics study and histological analysis confirmed the stability and minimal toxicity. In vitro anti-cancer cytotoxicity indicated a high potency of ADC with an IC50 value of <10 nM. In vivo anti-tumor efficacy showed that the anti-SSTR2 ADC with doses of 8 and 16 mg/kg body weight effectively inhibited tumor growth. This study demonstrated that the anti-SSTR2 ADC can target meningioma and reduce the tumor growth.

Complexes of the neurotensin receptor 1 with small-molecule ligands reveal structural determinants of full, partial, and inverse agonism.

Neurotensin receptor 1 (NTSR1) and related G protein-coupled receptors of the ghrelin family are clinically unexploited, and several mechanistic aspects of their activation and inactivation have remained unclear. Enabled by a new crystallization design, we present five new structures: apo-state NTSR1 as well as complexes with nonpeptide inverse agonists SR48692 and SR142948A, partial agonist RTI-3a, and the novel full agonist SRI-9829, providing structural rationales on how ligands modulate NTSR1. The inverse agonists favor a large extracellular opening of helices VI and VII, undescribed so far for NTSR1, causing a constriction of the intracellular portion. In contrast, the full and partial agonists induce a binding site contraction, and their efficacy correlates with the ability to mimic the binding mode of the endogenous agonist neurotensin. Providing evidence of helical and side-chain rearrangements modulating receptor activation, our structural and functional data expand the mechanistic understanding of NTSR1 and potentially other peptidergic receptors.

Anti-SSTR2 antibody-drug conjugate for neuroendocrine tumor therapy.

Neuroendocrine (NE) tumors include a diverse spectrum of hormone-secreting neoplasms that arise from the endocrine and nervous systems. Current chemo- and radio-therapies have marginal curative benefits. The goal of this study was to develop an innovative antibody-drug conjugate (ADC) to effectively treat NE tumors (NETs). First, we confirmed that somatostatin receptor 2 (SSTR2) is an ideal cancer cell surface target by analyzing 38 patient-derived NET tissues, 33 normal organs, and three NET cell lines. Then, we developed a new monoclonal antibody (mAb, IgG1, and kappa) to target two extracellular domains of SSTR2, which showed strong and specific surface binding to NETs. The ADC was constructed by conjugating the anti-SSTR2 mAb and antimitotic monomethyl auristatin E. In vitro evaluations indicated that the ADC can effectively bind, internalize, release payload, and kill NET cells. Finally, the ADC was evaluated in vivo using a NET xenograft mouse model to assess cancer-specific targeting, tolerated dosage, pharmacokinetics, and antitumor efficacy. The anti-SSTR2 ADC exclusively targeted and killed NET cells with minimal toxicity and high stability in vivo. This study demonstrates that the anti-SSTR2 ADC has a high-therapeutic potential for NET therapy.

Engineering an anti-HER2 biparatopic antibody with a multimodal mechanism of action.

The receptor tyrosine kinase HER2 acts as oncogenic driver in numerous cancers. Usually, the gene is amplified, resulting in receptor overexpression, massively increased signaling and unchecked proliferation. However, tumors become frequently addicted to oncogenes and hence are druggable by targeted interventions. Here, we design an anti-HER2 biparatopic and tetravalent IgG fusion with a multimodal mechanism of action. The molecule first induces HER2 clustering into inactive complexes, evidenced by reduced mobility of surface HER2. However, in contrast to our earlier binders based on DARPins, clusters of HER2 are thereafter robustly internalized and quantitatively degraded. This multimodal mechanism of action is found only in few of the tetravalent constructs investigated, which must target specific epitopes on HER2 in a defined geometric arrangement. The inhibitory effect of our antibody as single agent surpasses the combination of trastuzumab and pertuzumab as well as its parental mAbs in vitro and it is effective in a xenograft model.

Ablation of lncRNA Miat attenuates pathological hypertrophy and heart failure.

Rationale: The conserved long non-coding RNA (lncRNA) myocardial infarction associate transcript (Miat) was identified for its multiple single-nucleotide polymorphisms that are strongly associated with susceptibility to MI, but its role in cardiovascular biology remains elusive. Here we investigated whether Miat regulates cardiac response to pathological hypertrophic stimuli. Methods: Both an angiotensin II (Ang II) infusion model and a transverse aortic constriction (TAC) model were used in adult WT and Miat-null knockout (Miat-KO) mice to induce pathological cardiac hypertrophy. Heart structure and function were evaluated by echocardiography and histological assessments. Gene expression in the heart was evaluated by RNA sequencing (RNA-seq), quantitative real-time RT-PCR (qRT-PCR), and Western blotting. Primary WT and Miat-KO mouse cardiomyocytes were isolated and used in Ca2+ transient and contractility measurements. Results: Continuous Ang II infusion for 4 weeks induced concentric hypertrophy in WT mice, but to a lesser extent in Miat-KO mice. Surgical TAC for 6 weeks resulted in decreased systolic function and heart failure in WT mice but not in Miat-KO mice. In both models, Miat-KO mice displayed reduced heart-weight to tibia-length ratio, cardiomyocyte cross-sectional area, cardiomyocyte apoptosis, and cardiac interstitial fibrosis and a better-preserved capillary density, as compared to WT mice. In addition, Ang II treatment led to significantly reduced mRNA and protein expression of the Ca2+ cycling genes Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and ryanodine receptor 2 (RyR2) and a dramatic increase in global RNA splicing events in the left ventricle (LV) of WT mice, and these changes were largely blunted in Miat-KO mice. Consistently, cardiomyocytes isolated from Miat-KO mice demonstrated more efficient Ca2+ cycling and greater contractility. Conclusions: Ablation of Miat attenuates pathological hypertrophy and heart failure, in part, by enhancing cardiomyocyte contractility.

Distinct conformations of the HIV-1 V3 loop crown are targetable for broad neutralization.

The V3 loop of the HIV-1 envelope (Env) protein elicits a vigorous, but largely non-neutralizing antibody response directed to the V3-crown, whereas rare broadly neutralizing antibodies (bnAbs) target the V3-base. Challenging this view, we present V3-crown directed broadly neutralizing Designed Ankyrin Repeat Proteins (bnDs) matching the breadth of V3-base bnAbs. While most bnAbs target prefusion Env, V3-crown bnDs bind open Env conformations triggered by CD4 engagement. BnDs achieve breadth by focusing on highly conserved residues that are accessible in two distinct V3 conformations, one of which resembles CCR5-bound V3. We further show that these V3-crown conformations can, in principle, be attacked by antibodies. Supporting this conclusion, analysis of antibody binding activity in the Swiss 4.5 K HIV-1 cohort (n = 4,281) revealed a co-evolution of V3-crown reactivities and neutralization breadth. Our results indicate a role of V3-crown responses and its conformational preferences in bnAb development to be considered in preventive and therapeutic approaches.

Chaperone-assisted structure elucidation with DARPins.

Designed ankyrin repeat proteins (DARPins) are artificial binding proteins that have found many uses in therapy, diagnostics and biochemical research. They substantially extend the scope of antibody-derived binders. Their high affinity and specificity, rigidity, extended paratope, and facile bacterial production make them attractive for structural biology. Complexes with simple DARPins have been crystallized for a long time, but particularly the rigid helix fusion strategy has opened new opportunities. Rigid DARPin fusions expand crystallization space, enable recruitment of targets in a host lattice and reduce the size limit for cryo-EM. Besides applications in structural biology, rigid DARPin fusions also serve as molecular probes in cells to investigate spatial restraints in targets.