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@article{pmid42165126,

title = {A universal Fab targeting a conserved U1A-RNA epitope for RNA structure determination by cryo-EM},

author = {Ekaterina V Filippova and Daniel Krochmal and Somnath Mukherjee and Joseph A Piccirilli and Anthony A Kossiakoff},

doi = {10.1093/nar/gkag502},

issn = {1362-4962},

year  = {2026},

date = {2026-05-01},

urldate = {2026-05-01},

journal = {Nucleic Acids Res},

volume = {54},

number = {10},

abstract = {Recent advances in cryo-electron microscopy (cryo-EM) have made antigen-binding fragments (Fabs) essential tools in the field of structural biology. Fabs facilitate image alignment, thereby enhancing three-dimensional (3D) reconstruction, and increase the effective size of proteins, aiding in their structural elucidation. In this study, we sought to broaden the use of Fabs as fiducial markers to elucidate the structures of RNA molecules. Identifying an appropriate Fab for a specific RNA target can be particularly challenging due to RNA's inherent flexibility and tendency to assume multiple conformations, which complicate the process and prolong the structure determination timeline. To address this challenge, we designed a universal Fab that specifically recognizes a U1A-RNA epitope, thereby reducing the need for Fab selection tailored to each individual RNA target. We determined the cryo-EM structure of the class I ligase ribozyme complexed with a portable U1hpII loop bound to the U1A protein and the Fab. The resulting structure revealed that the Fab interacts with a conserved U1A-RNA binding region, which can be engineered into other RNA molecules. This strategy presents significant potential for streamlining the structural determination of various RNAs, which are essential for biological and biomedical research.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid41959119,

title = {Structural Basis of Serine Protease Inhibition by Antibodies from Biased Fab Phage-Display Libraries},

author = {Kyle J Anderson and Melody S Lee and Natalia Sevillano and Gang Chen and Michael J Hornsby and Sachdev S Sidhu and Charles S Craik},

doi = {10.64898/2026.03.12.711446},

issn = {2692-8205},

year  = {2026},

date = {2026-03-01},

urldate = {2026-03-01},

journal = {bioRxiv},

abstract = {Biased Fab phage-display libraries were designed to determine whether inhibitory CDR H3 motifs from potent anti-matriptase antibodies could be transferred to target homologous serine proteases. Using reverse-binding and substrate-like H3 motifs from parental clones A11 and E2 as templates, six synthetic libraries with 10 diversity were constructed. Selection against matriptase identified sixteen inhibitors with sub-100 nM potency, representing 100,000-fold improvement over circularized H3 loops alone. Selection against TMPRSS2, a serine protease implicated in viral entry and prostate cancer with 43% sequence identity to matriptase, yielded binders with micromolar inhibitory potency. Selection against urokinase plasminogen activator (uPA, 35% identity) identified binders that adopted a substrate-like CDR H3 binding mode in our structural models. Across all reference structures, including the separately identified uPA inhibitor AB2 (PDB: 9PYF, deposited with this work), benchmarking of five co-folding methods and rigid-body docking showed that co-folding consistently achieved acceptable to high quality DockQ scores, outperforming traditional docking and capturing the recognition of key active site determinants. Ensemble predictions of mutational binding energy changes (ΔΔ) using these models identified key paratope-epitope interactions, with predictions validated through mutagenesis. This work establishes a framework integrating biased antibody libraries with computational structure prediction and analysis for targeting conserved protease epitopes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid41875162,

title = {Dynamic translocation of Inside-Out proteins to the cell surface underlies cellular adaptation to cancer-induced stress},

author = {Tomasz Slezak and Kelly M O'Leary and Tanya Guevara Avella and Natalia Musial and Jinyang Li and Anna Andrzejczak and Elizabeth F Scott and Duc Anh Le and Anthony A Kossiakoff},

doi = {10.1073/pnas.2529493123},

issn = {1091-6490},

year  = {2026},

date = {2026-03-01},

urldate = {2026-03-01},

journal = {Proc Natl Acad Sci U S A},

volume = {123},

number = {13},

pages = {e2529493123},

abstract = {Inside-Out (I-O) protein display, the noncanonical surface localization of intracellular proteins, represents an underexplored feature of tumor cell biology. Here, we map the molecular landscape and trafficking mechanisms that control the presentation of I-O proteins on cancer cell membranes. Employing APEX2-mediated proximity biotinylation and a custom antibody generation and validation platform, we identified approximately 140 high-confidence I-O proteins, primarily ribosomal, proteasomal, chaperone, and translation factors, notably enriched in protein families associated with stress-response pathways. Validation of 500 antibodies encompassing 40 I-O targets across seven tumor cell lines confirmed selective and robust surface localization, while in vivo imaging in mouse xenografts demonstrated pronounced and tumor-specific antibody accumulation. I-O proteins were absent on peripheral blood mononuclear cells (PBMCs) and in normal tissues, indicating cancer cell selectivity. Functional analyses revealed that I-O protein tethering to the membrane is dependent on heparan sulfate interactions; enzymatic removal of these glycans led to the clearance of I-O proteins from the cell surface. Notably, the removed proteins returned to baseline levels within 6 h, indicating a dynamic balance related to Endoplasmic Reticulum (ER)-Golgi trafficking and cellular stress. Nearly half of these I-O proteins overlapped with known stress granule (SG) components; however, stress elements that promote SG formation do not similarly affect surface display of I-O proteins. Furthermore, I-O proteins are present on standard cancer cell lines under lower stress levels needed to induce SG formation, suggesting parallel yet mechanistically distinct aspects of the stress response. These findings position I-O display as a paradigm in protein trafficking, different from traditional secretion pathways and closely linked to stress response.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}