Biology subjects

Ghaffari-Kashani, S.

Publications and source records attributed to Ghaffari-Kashani, S..

3 recordsLinked to original sources

bioRxiv2026

Biophysical trade-offs in antibody evolution are resolved by conformation-mediated epistasis

Protein evolution is constrained by multidimensional biophysical factors, in which mutations that enhance one property often compromise another. Antibodies represent an extreme case: they evolve rapidly to bind diverse antigens, yet mutations that improve affinity can disrupt folding, reduce cell-surface trafficking, or promote self-reactivity, and are typically selected against during affinity maturation. Though biophysical characterization of individual antibodies suggests that such trade-offs are pervasive, their impact on antibody evolutionary trajectories remains unclear, in part because existing high-throughput biophysical methods rely on heterologous systems that are often poorly suited for human proteins. Here, we develop a high-throughput platform to quantify multiple biophysical parameters of large libraries of full-length proteins that are natively synthesized, processed, and displayed on human cells. We apply this approach to a human antibody lineage that matures to recognize divergent SARS-CoV-2 variants by measuring the surface expression, antigen affinity, and self-reactivity for all 213 possible evolutionary intermediates between the unmutated and mature sequences. These measurements reveal that mutations differentially affect these biophysical properties - in some cases, improving one property at the expense of another. We leverage these data to compute the likelihood of all possible evolutionary paths, finding that very few paths can navigate these multidimensional requirements. The few accessible paths acquire mutations in a specific order that either circumvent trade-offs between biophysical properties or offset deleterious effects on one property with beneficial effects on another. By determining the structures of the ancestral and evolved antibodies, we find that these coordinated mutational effects arise from a conformational rearrangement that alleviates steric clashes and reshapes the biophysical landscape, enabling otherwise inaccessible mutational paths. Together, this work defines the multidimensional biophysical constraints and structural mechanisms that govern antibody evolution and establishes a general framework for mapping and predicting the biophysical effects of mutations in human proteins.

evolutionary biology↗

bioRxiv2025

BTK Autoinhibition Analyzed by High-Throughput SH2 Domain Swaps

BTK, a Tec-family tyrosine kinase, resembles the Src and Abl kinases in that an SH2-SH3 module regulates the activity of the kinase domain, principally through an inhibitory interaction between the SH3 and kinase domains. In Src kinases, phosphorylation of a C-terminal tail latches the SH2 domain onto the kinase domain, positioning the SH3 domain in an inhibitory conformation; in Abl, interaction between the kinase domain and a myristoyl group on the N- terminal segment provides the same latching function. The structure of autoinhibited BTK resembles that of the Src and Abl kinases, but BTK lacks an SH2-kinase latch. To assess autoinhibition in BTK, we generated hundreds of chimeric BTK molecules and measured their fitness using a high-throughput assay in T and B cells. Surprisingly, many SH2 domains increased fitness when substituted into BTK. Analysis of one set of chimeric proteins demonstrated that the increase in fitness stems from the ability of the substituted SH2 domains to disrupt BTK autoinhibition while maintaining phosphotyrosine targeting. These results reveal the importance of distributed interactions between the SH2 and kinase domains of BTK in stabilizing the inhibitory conformation, and suggest that the specialized latching mechanisms in Src and Abl kinases may be later evolutionary refinements.

biochemistry↗

bioRxiv2023

Differential Requirement for Dimerization of the Membrane-Binding PH-TH Module of BTK in B cells and T cells

Brutons tyrosine kinase (BTK) is a major drug target in immune cells. The membrane-binding pleckstrin-homology and tec-homology (PH-TH) domains of BTK are required for signaling. In vitro, dimerization of the PH-TH module strongly stimulates BTK kinase activity. Whether BTK dimerizes in cells via the PH-TH module, and whether this dimerization is necessary for signaling, is unknown. To address this question, we developed high-throughput mutagenesis assays for BTK function in B cells and T cells. We measured the fitness costs for thousands of point mutations in the PH-TH module and kinase domain, allowing us to assess whether dimerization of the PH-TH module and BTK kinase activity are necessary for function. In Ramos B cells we find that neither PH-TH dimerization nor kinase activity is required for BTK signaling. Instead, in Ramos cells, BTK signaling is enhanced by mutations in the PH-TH module that increase membrane adsorption, even at the cost of reduced PH-TH dimerization. In contrast, in Jurkat T cells, we find that BTK signaling depends on both PH-TH dimerization and kinase activity. Evolutionary analysis shows that BTK proteins in fish and lower organisms, like all Tec kinases other than BTK, lack PH-TH dimerization but have active kinase domains. Thus, PH-TH dimerization is not intrinsically required for Tec-kinase activity, and is a special feature that evolved to exert stricter regulatory control on BTK kinase activity as adaptive immune systems gained increased complexity.

biochemistry↗