Andras Fiser

Predicting biologically relevant protein–protein interactions remains a major challenge in structural biology. Here, we present ProtLID2.0, a residue-specific pharmacophore framework that generates coordinate-based interaction preferences from molecular dynamics simulations of single-residue probes at receptor interfaces and uses these preferences to score docked complexes and identify cognate ligands. Benchmarking across 30 receptor systems and multiple docking methods demonstrated that ProtLID2.0 performs comparably to or better than HADDOCK for identifying near-native poses and ranking cognate ligands from decoy sets, while combining both scoring functions consistently improved performance. These results establish ProtLID2.0 as a robust and complementary scoring framework for protein–protein docking and ligand identification.

The human immunoglobulin superfamily (IgSF) encompasses hundreds of proteins involved in cell–cell adhesion, neural connectivity, junctional organization, and immune regulation, with many serving as key checkpoint proteins. To elucidate the evolutionary history of human IgSF members, we systematically analyzed all available eukaryotic reference genomes to determine when each IgSF subfamily first appeared. The human IgSF was partitioned into six major evolutionary timeframes: Metazoa, Vertebrata, Gnathostomata, Tetrapoda, Amniota, and Mammalia. Although proteins were grouped solely by their conservation across eukaryotes, their biological functions clustered naturally, reflecting how new physiological systems create selective pressures that drive the appearance, retention, and diversification of protein architectures suited to those functions. Conservation and functional analyses indicate that human IgSF members arising in tetrapods and amniotes primary regulate the strength and duration of immune responses and form many of the critical components of the immune synapse, while IgSF genes that appear first in mammals have evolved to fine-tune immune activation thresholds to support maternal–fetal tolerance. Case studies are provided to illustrate three key evolutionary themes: (i) highly conserved yet catalytically inactive proteins retain essential regulatory functions, (ii) functional convergence among evolutionarily distinct IgSF families, and (iii) compensatory evolution within adaptive immunity following a lineage-specific loss of an IgSF member. Together, these findings establish an evolutionary framework for organizing the human IgSF by both ancestry and function, providing a foundation for assessing IgSF importance. Notably, this study facilitates the identification of conserved but understudied proteins that emerged alongside the development of the adaptive immune system, highlighting them as promising candidates for future experimental investigation.

Supplementary Data and figure legends

Supplementary Figures and Legends

Single-domain antibodies, such as variable heavy domain of heavy chain (VHH) domains from camelids, are an attractive platform for therapeutic purposes. VHHs have a smaller size than traditional antibodies, which harbors certain advantages such as tissue penetration or accessing epitopes that may be shielded by protein domains. However, the smaller size of VHHs typically involves a smaller binding site, which can limit the scope of related antigens they are able to bind and, in the specific case of antiviral VHHs, render them susceptible to escape by a single point mutation. Here, we present a combined computational and experimental protein engineering approach to broaden the reactivity of SARS-CoV-1 receptorbinding domain (RBD)-specific VHH-72 for SARS-CoV-2 and Delta. Our strategy focuses on increasing the size of the binding site by imparting “second site” interactions toward heterologous antigens. We utilized the residue-based pharmacophore modeling approach, Protein-ligandinterface design (ProtLID), to identify potential productive side chain interactions in this second site and then encoded the ProtLID-predicted restricted diversity into a VHH-72-based phage library, which we then screened for cross-reactive SARS-CoV-1/2 RBD clones. Based on sequence analysis from the resulting population of functional VHH clones, we identified a VHH-72 triple mutant (T60P.D61P.D100eY), which maintained high affinity for SARS-CoV-1 RBD but was enhanced by 18- and 20-fold for RBDs from SARS-CoV-2 and Delta relative to WT VHH-72. These results highlight the potential of computationally customizing phage display diversity for single-domain binding proteins and provide a strategy for designing extension of protein binding interfaces.

Background: Schwann cells (SCs) and their plasticity contribute to the peripheral nervous system’s capacity for nerve regeneration after injury. The Egr2/Krox20 promoter antisense RNA (Egr2-AS) recruits chromatin remodeling complexes to inhibit Egr2 transcription following peripheral nerve injury. Methods: RNA-seq and ATAC-seq were performed on control cells, Lenti-GFP-transduced cells, and cells overexpressing Egr2-AS (Lenti-AS). Egr2 AS-RNA was cloned into the pLVX-DsRed-Express2-N1 lentiviral expression vector (Clontech, Mountain View, CA, USA), and the levels of AS-RNA expression were determined. Ezh2 and Wdr5 were immunoprecipitated from rat SCs and RT-qPCR was performed against AS-Egr2 RNA. ChIP followed by DNA purification columns was used to perform qPCR for relevant promoters. Hi-C, HiC-DC+, R, Bioconductor, and TOBIAS were used for significant and differential loop analysis, identifications of COREs and CORE-promotor loops, comparisons of TF activity at promoter sites, and identification of site-specific TF footprints. OnTAD was used to detect TADs, and Juicer was used to identify A/B compartments. Results: Here we show that a Neuregulin-ErbB2/3 signaling axis mediates binding of the Egr2-AS to YY1^Ser184 and regulates its expression. Egr2-AS modulates the chromatin accessibility of Schwann cells and interacts with two distinct histone modification complexes. It binds to EZH2 and WDR5 and enables targeting of H3K27me3 and H3K4me3 to promoters of Egr2 and C-JUN, respectively. Expression of the Egr2-AS results in reorganization of the global chromatin landscape and quantitative changes in the loop formation and contact frequency at domain boundaries exhibiting enrichment for AP-1 genes. In addition, the Egr2-AS induces changes in the hierarchical TADs and increases transcription factor binding scores on an inter-TAD loop between a super-enhancer regulatory hub and the promoter of mTOR. Conclusions: Our results show that Neuregulin-ErbB2/3-YY1 regulates the expression of Egr2-AS, which mediates remodeling of the chromatin landscape in Schwann cells.