Biology subjects

Binder, S. C.

Publications and source records attributed to Binder, S. C..

6 recordsLinked to original sources

bioRxiv2025

Multiple broadly neutralizing antibody lineages can co-exist and mature in the same germinal centres

An efficacious HIV vaccine will need to generate broadly neutralizing antibodies (bnAbs) against distinct viral epitopes. To facilitate this, immunogens targeting precursor B cells of bnAbs have been developed. With this strategy, individual immunogens can even target multiple lineages, thereby beneficially limiting the number of immunogens needed for a multi-bnAb generating vaccine. However, it is unclear whether this diminishes responses compared to isolated targeting of lineages with distinct immunogens. Here, we address this using an in silico model of naive B cell activation and affinity maturation in germinal centres. By incorporating (i) precursor properties and (ii) epitope masking by antibodies obtained from germinal centre-derived plasma cells, the model recapitulated features of bnAb lineage evolution as seen in pre-clinical mouse models. Subsequent model analysis suggested that under physiologically relevant conditions, priming of multiple bnAb lineages with a single immunogen was additive, thus, supporting the development of vaccines that target multiple lineages.

immunology↗

bioRxiv2025

Development of AI-designed protein binders for detection and targeting of cancer cell surface proteins

Artificial intelligence (AI)-based protein design opens new avenues for the rapid generation of new research tools and therapeutics, but experimental validation lags behind the computational design throughput. Here, we present a scalable workflow for the discovery and validation of AI-designed protein binders. Leveraging the RFdiffusion protein design pipeline with a custom filter for stable alpha-helical bundle folds, we construct libraries of thousands of AI-binders against cancer-associated surface proteins. Mammalian cell-surface and phage display screening yield multiple high-affinity PD-L1 binders but fewer hits for CD276 (B7-H3) and VTCN1 (B7-H4), reflecting the target-dependent efficiency of RFdiffusion in generating high-quality designs. Using our experimentally validated AI-designed binder libraries, we benchmark freely available structure prediction models. We find that interface predicted template modelling (ipTM) scores by Chai-1 with ESM embedding correlate well with experimental success and even predict deleterious effects of binding interface mutations. To demonstrate the versatility of AI-binders as research tools, we deploy them in CAR-T cells and also assemble them with fluorophore-labeled streptavidin into tetravalent quattrobinders, which achieve antibody-comparable staining of endogenous PD-L1 by flow cytometry. With high production yields and accessible structural models, AI-designed quattrobinders are versatile and cost-effective research tools amenable to community-driven validation and optimization.

bioengineering↗

bioRxiv2024

The SAVED domain of the type III CRISPR protease CalpL is a ring nuclease

Prokaryotic CRISPR-Cas immune systems detect and cleave foreign nucleic acids. In type III CRISPR-Cas systems, the Cas10 subunit of the activated recognition complex synthesizes cyclic oligoadenylates (cOAs), second messengers that activate downstream ancillary effector proteins. Once the viral attack has been weathered, elimination of extant cOA is essential to limit the antiviral response and to allow cellular recovery. Various families of ring nucleases have been identified, specializing in the degradation of cOAs either as standalone enzymes or as domains of effector proteins. Here we describe the ring nuclease activity inherent in the SAVED domain of the cA4-activated CRISPR Lon protease CalpL. We characterize the kinetics of cA4 cleavage and identify key catalytic residues. We demonstrate that cA4-incuced oligomerization of CalpL is essential not only for activation of the protease, but is also required for nuclease activity. Further, the nuclease activity of CalpL poses a limitation to the protease reaction, indicating a mechanism for regulation of the CalpL/T/S signaling cascade. This work is the first demonstration of a catalytic SAVED domain and gives new insights into the dynamics of transcriptional adaption in CRISPR defense systems which are not aimed at abortive infection but rather at a reversible adaption to phage attack.

biochemistry↗

bioRxiv2024

Structural basis of Cdk7 activation by dual T-loop phosphorylation

Cyclin-dependent kinase 7 (Cdk7) occupies a central position in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of the general transcription factor TFIIH. Cdk7 forms an active complex upon association with Cyclin H and Mat1, and its catalytic activity is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the fully activated human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates a set of basic residues conserved in other CDKs, pS164 nucleates an arginine network involving all three subunits that is unique to the ternary Cdk7 complex. We identify differential dependencies of kinase activity and substrate recognition on individual phosphorylations within the Cdk7 T loop. The CAK function of Cdk7 is not affected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by phosphorylation at T170. Moreover, dual T-loop phosphorylation at both T170 and S164 stimulates multi-site phosphorylation of transcriptional substrates--the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and the SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7-regulatory phosphorylation is a two-step process in which phosphorylation of S164 precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing Cdk7 processivity, while the canonical pT170 enhances kinase activity towards critical substrates involved in transcription.

biochemistry↗

bioRxiv2023

Antagonistic nanobodies reveal mechanism of GSDMD pore formation and unexpected therapeutic potential

Activation of various inflammasomes converges on the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases, followed by oligomerization of the N-terminal domain (GSDMDNT) and the assembly of pores penetrating target membranes. Yet, it remained unclear what triggers the conformational changes that allow membrane insertion, as methods to study pore formation in living cells were limited. We raised nanobodies specific for human GSDMD and found two nanobodies that prevent pyroptosis and IL-1{beta} release when expressed in the cytosol of human macrophages. Nanobody binding to GSDMDNT blocked its oligomerization, while inflammasome assembly and GSDMD processing itself were not affected. The nanobody-stabilized monomers of GSDMDNT partitioned into the plasma membrane, suggesting that pore formation is initiated by insertion of monomers, followed by oligomerization in the target membrane. When GSDMD pore formation was inhibited, cells still underwent caspase-1-dependent apoptosis, likely due to the substantially augmented caspase-1 activity. This hints at a novel layer of regulation of caspase-1 activity by GSDMD pores. Moreover, we revealed the unexpected therapeutic potential of antagonistic GSDMD nanobodies, as recombinant nanobodies added to the medium prevented cell death by pyroptosis, likely by entering through GSDMD pores and curtailing the assembly of additional pores. GSDMD nanobodies may thus be suitable to treat the ever-growing list of diseases caused by activation of the (non-) canonical inflammasomes.

immunology↗

bioRxiv2020

Rate of immune complex cycling in follicular dendritic cell determines the extent of protecting antigen integrity and availability to germinal center B cells

Follicular Dendritic Cells (FDCs) retain immune complexes (ICs) for prolonged time periods and are important for germinal center (GC) reactions. ICs undergo periodic cycling in FDCs, a mechanism supporting an extended half-life of antigen. Based on experimental data we estimated that the average residence time of Phycoerythrin-ICs (PE-ICs) on FDC surface and interior were 21 and 36 minutes, respectively. GC simulations show that antigen cycling might impact GC dynamics due to redistribution of antigen on the FDC surface and by protecting antigen from degradation. Antigen protection and influence on GC dynamics varied with antigen cycling time and total antigen concentration. Simulations predict that blocking antigen cycling terminates the GC reaction and decreases plasma cell production. Considering that cycling of antigen could be a target for the modulation of GC reactions, our findings highlight the importance of understanding the mechanism and regulation of IC cycling in FDCs.

immunology↗