Publications

@article{pmid40489625,

title = {Conformation-specific synthetic intrabodies modulate mTOR signaling with subcellular spatial resolution},

author = {Kelly M O'Leary and Tomasz Slezak and Anthony A Kossiakoff},

doi = {10.1073/pnas.2424679122},

issn = {1091-6490},

year  = {2025},

date = {2025-06-01},

urldate = {2025-06-01},

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

volume = {122},

number = {24},

pages = {e2424679122},

abstract = {Subcellular compartmentalization is integral to the spatial regulation of mechanistic target of rapamycin (mTOR) signaling. However, the biological outputs associated with location-specific mTOR signaling events are poorly understood and challenging to decouple. Here, we engineered synthetic intracellular antibodies (intrabodies) that are capable of modulating mTOR signaling with genetically programmable spatial resolution. Epitope-directed phage display was exploited to generate high affinity synthetic antibody fragments (Fabs) against the FKBP12-Rapamycin binding site of mTOR (mTOR). We determined high-resolution crystal structures of two unique Fabs that discriminate distinct conformational states of mTOR through recognition of its substrate recruitment interface. By leveraging these conformation-specific binders as intracellular probes, we uncovered the structural basis for an allosteric mechanism governing mTOR complex 1 (mTORC1) stability mediated by subtle structural adjustments within mTOR. Furthermore, our results demonstrated that synthetic binders emulate natural substrates by employing divergent yet complementary hydrophobic residues at defined positions, underscoring the broad molecular recognition capability of mTOR. Intracellular signaling studies showed differential time-dependent inhibition of S6 kinase 1 and Akt phosphorylation by genetically encoded intrabodies, thus supporting a mechanism of inhibition analogous to the natural product rapamycin. Finally, we implemented a feasible approach to selectively modulate mTOR signaling in the nucleus through spatially programmed intrabody expression. These findings establish intrabodies as versatile tools for dissecting the conformational regulation of mTORC1 and should be useful to explore how location-specific mTOR signaling influences disease progression.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40411427,

title = {Synthetic antibodies targeting EphA2 induce diverse signaling-competent clusters with differential activation},

author = {Jarrett J Adams and Heather A Bruce and Suryasree Subramania and Lynda Ploder and Julia Garcia and Isabelle Pot and Levi L Blazer and Alexander U Singer and Sachdev S Sidhu},

doi = {10.1002/pro.70145},

issn = {1469-896X},

year  = {2025},

date = {2025-06-01},

urldate = {2025-06-01},

journal = {Protein Sci},

volume = {34},

number = {6},

pages = {e70145},

abstract = {The receptor tyrosine kinase EphA2 interacts with ephrin (Efn) ligands to mediate bi-directional signals that drive cellular sorting processes during tissue development. In the context of various cancers, EphA2 can also drive invasive metastatic disease and represents an important target for cancer therapeutics. Natural Efn ligands sterically seed intertwined EphA2 clusters capable of recruiting intracellular kinases to mediate trans-phosphorylation. Synthetic proteins, such as antibodies (Abs), can mimic Efn ligands to trigger EphA2 signaling, leading to receptor internalization and degradation, and enabling intracellular delivery of conjugated drugs. Furthermore, Abs are capable of recruiting EphA2 into clusters distinct from those seeded by Efn. We developed three synthetic Abs targeting distinct EphA2 domains and determined the paratope valency necessary for agonist or antagonist properties of each of the three epitopes. Structural modeling of monovalent Fabs in complex with EphA2 elucidated competitive and non-competitive mechanisms of inhibition of EphA2 canonical signaling. Likewise, modeling of clusters induced by bivalent IgGs elucidated multiple signaling-competent EphA2 clusters capable of triggering a continuum of signaling strengths and provided insights into the requirement for multimerization of EphA2 to trigger phosphorylation. Our study shows how different agonist clusters lead to distinct kinase recruitment efficiencies to modify phosphotyrosine signal strength, and provides a panel of anti-EphA2 Abs as reagents for the development of therapeutics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40384600,

title = {Molecular characterization of a synthetic neutralizing antibody targeting p67 of Theileria parva},

author = {Shane Miersch and Alex U Singer and Chao Chen and Fred Fellouse and Anupriya Gopalsamy and Luciano Silva E Costa and Anna Lacasta and Hannah Chege and Naomi Chege and Vishvanath Nene and Sachdev S Sidhu},

doi = {10.1002/pro.70153},

issn = {1469-896X},

year  = {2025},

date = {2025-06-01},

urldate = {2025-06-01},

journal = {Protein Sci},

volume = {34},

number = {6},

pages = {e70153},

abstract = {The Theileria parva sporozoite surface antigen p67 is a target of the bovine humoral immune response that generates antibodies capable of providing protection against subsequent infection. As a result, p67 has been the subject of efforts aimed at the development of an anti-sporozoite subunit vaccine. Previous studies have identified neutralizing epitopes in the N- and C-terminal regions of the full-length protein and shown that immunization with a C-terminal fragment of p67 (p67C) alone is capable of eliciting protection. To identify additional neutralizing epitopes in p67C, selections were conducted against it using a phage-displayed synthetic antibody library. An antibody that neutralized the sporozoite in vitro was identified, and the crystal structure of a Fab:peptide complex was elucidated. Mutagenesis studies aimed at validating and further characterizing the Fab:peptide interaction identified critical residues involved in binding and neutralization. This study also validates distinct epitopes for previously reported neutralizing antibodies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40329529,

title = {Locking CBL TKBD in its native conformation presents a novel therapeutic opportunity in mutant CBL-dependent leukemia},

author = {Syed Feroj Ahmed and Jayanthi Anand and Wei Zhang and Lori Buetow and Loveena Rishi and Louise Mitchell and Jonathan Bohlen and Sergio Lilla and Gary J Sibbet and Colin Nixon and Amrita Patel and Karolina A Majorek and Sara Zanivan and Jacinta C Bustamante and Sachdev S Sidhu and Karen Blyth and Danny T Huang},

doi = {10.1016/j.ymthe.2025.04.042},

issn = {1525-0024},

year  = {2025},

date = {2025-05-01},

urldate = {2025-05-01},

journal = {Mol Ther},

abstract = {Casitas B-lineage lymphoma (CBL) is an E3 ubiquitin ligase critical for negatively regulating receptor protein tyrosine kinases (RTKs). Deleterious CBL mutants lose E3 activity, but act as adaptors that gain function to cause myeloproliferative neoplasms. Currently, there is no targeted treatment available for patients with CBL mutant-dependent disorders. By combining phage-display technology and structure-based optimization, we discovered CBLock, a nanomolar affinity peptide inhibitor, that binds the substrate-binding site of CBL's tyrosine kinase binding domain (TKBD). CBLock disrupts the interaction between CBL mutants and RTKs, thereby impairing RTK-mediated priming of adaptor function of CBL mutants and downstream signaling. Notably, CBLock binds TKBD without inducing conformational changes, thereby preserving its ligand-free native conformation. In contrast, when CBL binds RTK substrates, TKBD undergoes a conformational change. Maintaining the native CBL TKBD conformation was crucial for CBLock to inhibit proliferation, induce cell-cycle arrest, and promote apoptosis in leukemia cells harboring CBL mutations. In a mouse xenograft model of acute myeloid leukemia (AML), CBLock reduced tumor burden and improved survival rate. Moreover, CBLock inhibited the proliferation of cells derived from patients with CBL mutations. Therefore, inhibiting CBL TKBD in its native state presents a promising therapeutic opportunity in targeting mutant CBL-dependent leukemia.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40402577,

title = {High affinity CD16 polymorphism associated with reduced risk of severe COVID-19},

author = {Anita E Qualls and Tasha Tsao and Irene Lui and Shion A Lim and Yapeng Su and Ernie Chen and Dylan Duchen and Holden T Maecker and Seunghee Kim-Schulze and Ruth R Montgomery and Florian Krammer and Charles R Langelier and Ofer Levy and Lindsey R Baden and Esther Melamed and Lauren Ir Ehrlich and Grace A McComsey and Rafick P Sekaly and Charles B Cairns and Elias K Haddad and Albert C Shaw and David A Hafler and David B Corry and Farrah Kheradmand and Mark A Atkinson and Scott C Brakenridge and Nelson I Agudelo Higuita and Jordan P Metcalf and Catherine L Hough and William B Messer and Bali Pulendran and Kari C Nadeau and Mark M Davis and Ana Fernandez-Sesma and Viviana Simon and Monica Kraft and Christian Bime and Carolyn S Calfee and David J Erle and Joanna Schaenmann and Al Ozonoff and Bjoern Peters and Steven H Kleinstein and Alison D Augustine and Joann Diray-Arce and Patrice M Becker and Nadine Rouphael and Impacc Network and Jason D Goldman and Daniel R Calabrese and James R Heath and James A Wells and Elaine F Reed and Lewis L Lanier and Harry Pickering and Oscar A Aguilar},

doi = {10.1172/jci.insight.191314},

issn = {2379-3708},

year  = {2025},

date = {2025-05-01},

urldate = {2025-05-01},

journal = {JCI Insight},

abstract = {CD16 is an activating Fc receptor on natural killer cells that mediates antibody-dependent cellular cytotoxicity (ADCC), a key mechanism in antiviral immunity. However, the role of NK cell-mediated ADCC in SARS-CoV-2 infection remains unclear, particularly whether it limits viral spread and disease severity or contributes to the immunopathogenesis of COVID-19. We hypothesized that the high-affinity CD16AV176 polymorphism influences these outcomes. Using a novel in vitro reporter system, we demonstrated that CD16AV176 is a more potent and sensitive activator than the common CD16AF176 allele. To assess its clinical relevance, we analyzed 1,027 hospitalized COVID-19 patients from the Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC), a comprehensive longitudinal dataset with extensive transcriptomic, proteomic, and clinical data. The high-affinity CD16AV176 allele was associated with a significantly reduced risk of ICU admission, mechanical ventilation, and severe disease trajectories. Lower anti-SARS-CoV-2 IgG titers were correlated to CD16AV176; however, there was no difference in viral load across CD16 genotypes. Proteomic analysis revealed that participants homozygous for CD16AV176 had lower levels of inflammatory mediators. These findings suggest that CD16AV176 enhances early NK cell-mediated immune responses, limiting severe respiratory complications in COVID-19. This study identifies a protective genetic factor against severe COVID-19, informing future host-directed therapeutic strategies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40399676,

title = {Effect of phosphorylation barcodes on arrestin binding to a chemokine receptor},

author = {Qiuyan Chen and Christopher T Schafer and Somnath Mukherjee and Kai Wang and Martin Gustavsson and James R Fuller and Katelyn Tepper and Thomas D Lamme and Yasmin Aydin and Parth Agrawal and Genki Terashi and Xin-Qiu Yao and Daisuke Kihara and Anthony A Kossiakoff and Tracy M Handel and John J G Tesmer},

doi = {10.1038/s41586-025-09024-9},

issn = {1476-4687},

year  = {2025},

date = {2025-05-01},

urldate = {2025-05-01},

journal = {Nature},

abstract = {Unique phosphorylation 'barcodes' installed in different regions of an active seven-transmembrane receptor by different G-protein-coupled receptor (GPCR) kinases (GRKs) have been proposed to promote distinct cellular outcomes, but it is unclear whether or how arrestins differentially engage these barcodes. Here, to address this, we developed an antigen-binding fragment (Fab7) that recognizes both active arrestin2 (β-arrestin1) and arrestin3 (β-arrestin2) without interacting with bound receptor polypeptides. We used Fab7 to determine the structures of both arrestins in complex with atypical chemokine receptor 3 (ACKR3) phosphorylated in different regions of its C-terminal tail by either GRK2 or GRK5 (ref. ). The GRK2-phosphorylated ACKR3 resulted in more heterogeneous 'tail-mode' assemblies, whereas phosphorylation by GRK5 resulted in more rigid 'ACKR3-adjacent' assemblies. Unexpectedly, the finger loops of both arrestins engaged the micelle surface rather than the receptor intracellular pocket, with arrestin3 being more dynamic, partly because of its lack of a membrane-anchoring motif. Thus, both the region of the barcode and the arrestin isoform involved can alter the structure and dynamics of GPCR-arrestin complexes, providing a possible mechanistic basis for unique downstream cellular effects, such as the efficiency of chemokine scavenging and the robustness of arrestin binding in ACKR3.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40312771,

title = {Hypoxia Induces Extensive Protein and Proteolytic Remodeling of the Cell Surface in Pancreatic Adenocarcinoma (PDAC) Cell Lines},

author = {Irene Lui and Kaitlin Schaefer and Lisa L Kirkemo and Jie Zhou and Rushika M Perera and Kevin K Leung and James A Wells},

doi = {10.1021/acs.jproteome.4c01037},

issn = {1535-3907},

year  = {2025},

date = {2025-05-01},

urldate = {2025-05-01},

journal = {J Proteome Res},

abstract = {The tumor microenvironment (TME) plays a crucial role in cancer progression. Hypoxia is a hallmark of the TME and induces a cascade of molecular events that affect cellular processes involved in metabolism, metastasis, and proteolysis. In pancreatic ductal adenocarcinoma (PDAC), tumor tissues are extremely hypoxic. Here, we leveraged mass spectrometry technologies to examine hypoxia-induced alterations in the abundance and proteolytic modifications to cell surface and secreted proteins. Across four PDAC cell lines, we discovered extensive proteolytic remodeling of cell surface proteins involved in cellular adhesion and motility. Looking outward at the surrounding secreted space, we identified hypoxia-regulated secreted and proteolytically shed proteins involved in regulating the humoral immune and inflammatory response, and an upregulation of proteins involved in metabolic processing and tissue development. Combining cell surface N-terminomics and secretomics to evaluate the cellular response to hypoxia enabled us to identify significantly altered candidate proteins which may serve as potential biomarkers and therapeutic targets in PDAC. Furthermore, this approach provides a blueprint for studying dysregulated extracellular proteolysis in other cancers and inflammatory diseases.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chen2025,

title = {Structural basis for nucleolin recognition of MYC promoter G-quadruplex},

author = {Luying Chen and Jonathan Dickerhoff and Ke-wei Zheng and Satchal Erramilli and Hanqiao Feng and Guanhui Wu and Buket Onel and Yuwei Chen and Kai-Bo Wang and Megan Carver and Clement Lin and Saburo Sakai and Jun Wan and Charles Vinson and Laurence Hurley and Anthony A. Kossiakoff and Nanjie Deng and Yawen Bai and Nicholas Noinaj and Danzhou Yang},

doi = {10.1126/science.adr1752},

issn = {1095-9203},

year  = {2025},

date = {2025-04-18},

urldate = {2025-04-18},

journal = {Science},

volume = {388},

number = {6744},

publisher = {American Association for the Advancement of Science (AAAS)},

abstract = {The MYC oncogene promoter G-quadruplex (MycG4) regulates transcription and is a prevalent G4 locus in immortal cells. Nucleolin, a major MycG4-binding protein, exhibits greater affinity for MycG4 than for nucleolin recognition element (NRE) RNA. Nucleolin’s four RNA binding domains (RBDs) are essential for high-affinity MycG4 binding. We present the 2.6-angstrom crystal structure of the nucleolin-MycG4 complex, revealing a folded parallel three-tetrad G-quadruplex with two coordinating potassium ions (K+), interacting with RBD1, RBD2, and Linker12 through its 6–nucleotide (nt) central loop and 5′ flanking region. RBD3 and RBD4 bind MycG4’s 1-nt loops as demonstrated by nuclear magnetic resonance (NMR). Cleavage under targets and tagmentation sequencing confirmed nucleolin’s binding to MycG4 in cells. Our results revealed a G4 conformation-based recognition by a regulating protein through multivalent interactions, suggesting that G4s are nucleolin’s primary cellular substrates, indicating G4 epigenetic transcriptional regulation and helping G4-targeted drug discovery.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ramesh2025,

title = {Mapping the extracellular molecular architecture of the pAg-signaling complex with α-Butyrophilin antibodies},

author = {Amrita Ramesh and Sobhan Roy and Tomasz Slezak and James Fuller and Hortencia Graves and Murad R. Mamedov and Alexander Marson and Anthony A. Kossiakoff and Erin J. Adams},

doi = {10.1038/s41598-025-94347-w},

issn = {2045-2322},

year  = {2025},

date = {2025-04-09},

urldate = {2025-12-00},

journal = {Sci Rep},

volume = {15},

number = {1},

publisher = {Springer Science and Business Media LLC},

abstract = {Target cells trigger Vγ9Vδ2 T cell activation by signaling the intracellular accumulation of phospho-antigen metabolites (pAgs) through Butyrophilin (BTN)-3A1 and BTN2A1 to the Vγ9Vδ2 T cell receptor (TCR). An incomplete understanding of the molecular dynamics in this signaling complex hampers Vγ9Vδ2 T cell immunotherapeutic efficacy. A panel of engineered α-BTN3A1 and α-BTN2A1 antibody (mAb) reagents was used to probe the roles of BTN3A1 and BTN2A1 in pAg signaling. Modified α-BTN3A1 mAbs with increased inter-Fab distances establish that tight clustering of BTN3A1 is not necessary to stimulate Vγ9Vδ2 T cell activation, and that antagonism may occur through occlusion of a critical binding interaction between BTN3A1 and a yet unknown co-receptor. Finally, a panel of additional α-BTN2A1 antagonists utilize different biophysical mechanisms to compete with Vγ9Vδ2 TCRs for BTN2A1 binding. The complex structures of BTN2A1 ectodomain and Fabs from three antagonist mAbs provide molecular insights into BTN2A1 epitopes critical for pAg-signaling.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40178992,

title = {Engineered Proteins and Chemical Tools to Probe the Cell Surface Proteome},

author = {Kevin K Leung and Kaitlin Schaefer and Zhi Lin and Zi Yao and James A Wells},

doi = {10.1021/acs.chemrev.4c00554},

issn = {1520-6890},

year  = {2025},

date = {2025-04-01},

urldate = {2025-04-01},

journal = {Chem Rev},

abstract = {The cell surface proteome, or surfaceome, is the hub for cells to interact and communicate with the outside world. Many disease-associated changes are hard-wired within the surfaceome, yet approved drugs target less than 50 cell surface proteins. In the past decade, the proteomics community has made significant strides in developing new technologies tailored for studying the surfaceome in all its complexity. In this review, we first dive into the unique characteristics and functions of the surfaceome, emphasizing the necessity for specialized labeling, enrichment, and proteomic approaches. An overview of surfaceomics methods is provided, detailing techniques to measure changes in protein expression and how this leads to novel target discovery. Next, we highlight advances in proximity labeling proteomics (PLP), showcasing how various enzymatic and photoaffinity proximity labeling techniques can map protein-protein interactions and membrane protein complexes on the cell surface. We then review the role of extracellular post-translational modifications, focusing on cell surface glycosylation, proteolytic remodeling, and the secretome. Finally, we discuss methods for identifying tumor-specific peptide MHC complexes and how they have shaped therapeutic development. This emerging field of neo-protein epitopes is constantly evolving, where targets are identified at the proteome level and encompass defined disease-associated PTMs, complexes, and dysregulated cellular and tissue locations. Given the functional importance of the surfaceome for biology and therapy, we view surfaceomics as a critical piece of this quest for neo-epitope target discovery.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40187349,

title = {Structural basis of BAK sequestration by MCL-1 in apoptosis},

author = {Shagun Srivastava and Giridhar Sekar and Adedolapo Ojoawo and Anup Aggarwal and Elisabeth Ferreira and Emiko Uchikawa and Meek Yang and Christy R Grace and Raja Dey and Yi-Lun Lin and Cristina D Guibao and Seetharaman Jayaraman and Somnath Mukherjee and Anthony A Kossiakoff and Bin Dong and Alexander Myasnikov and Tudor Moldoveanu},

doi = {10.1016/j.molcel.2025.03.013},

issn = {1097-4164},

year  = {2025},

date = {2025-03-01},

urldate = {2025-03-01},

journal = {Mol Cell},

abstract = {Apoptosis controls cell fate, ensuring tissue homeostasis and promoting disease when dysregulated. The rate-limiting step in apoptosis is mitochondrial poration by the effector B cell lymphoma 2 (BCL-2) family proteins BAK and BAX, which are activated by initiator BCL-2 homology 3 (BH3)-only proteins (e.g., BIM) and inhibited by guardian BCL-2 family proteins (e.g., MCL-1). We integrated structural, biochemical, and pharmacological approaches to characterize the human prosurvival MCL-1:BAK complex assembled from their BCL-2 globular core domains. We reveal a canonical interaction with BAK BH3 bound to the hydrophobic groove of MCL-1 and disordered and highly dynamic BAK regions outside the complex interface. We predict similar conformations of activated effectors in complex with other guardians or effectors. The MCL-1:BAK complex is a major cancer drug target. We show that MCL-1 inhibitors are inefficient in neutralizing the MCL-1:BAK complex, requiring high doses to initiate apoptosis. Our study underscores the need to design superior clinical candidate MCL-1 inhibitors.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40060520,

title = {β-Amyloid Induces Microglial Expression of GPC4 and APOE Leading to Increased Neuronal Tau Pathology and Toxicity},

author = {Brandon B Holmes and Thaddeus K Weigel and Jesseca M Chung and Sarah K Kaufman and Brandon I Apresa and James R Byrnes and Kaan S Kumru and Jaime Vaquer-Alicea and Ankit Gupta and Indigo V L Rose and Yun Zhang and Alissa L Nana and Dina Alter and Lea T Grinberg and Salvatore Spina and Kevin K Leung and Carlo Condello and Martin Kampmann and William W Seeley and Jaeda C Coutinho-Budd and James A Wells},

doi = {10.1101/2025.02.20.637701},

issn = {2692-8205},

year  = {2025},

date = {2025-02-01},

urldate = {2025-02-01},

journal = {bioRxiv},

abstract = {To elucidate the impact of Aβ pathology on microglia in Alzheimer's disease pathogenesis, we profiled the microglia surfaceome following treatment with Aβ fibrils. Our findings reveal that Aβ-associated human microglia upregulate Glypican 4 (GPC4), a GPI-anchored heparan sulfate proteoglycan (HSPG). In a  amyloidosis model, glial GPC4 expression exacerbates motor deficits and reduces lifespan, indicating that glial GPC4 contributes to a toxic cellular program during neurodegeneration. In cell culture, GPC4 enhances microglia phagocytosis of tau aggregates, and shed GPC4 can act  to facilitate tau aggregate uptake and seeding in neurons. Additionally, our data demonstrate that GPC4-mediated effects are amplified in the presence of APOE. These studies offer a mechanistic framework linking Aβ and tau pathology through microglial HSPGs and APOE.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39982408,

title = {De Novo Design of Proteins That Bind Naphthalenediimides, Powerful Photooxidants with Tunable Photophysical Properties},

author = {Samuel I Mann and Zhi Lin and Sophia K Tan and Jiaqi Zhu and Zachary X W Widel and Ian Bakanas and Jarrett P Mansergh and Rui Liu and Mark J S Kelly and Yibing Wu and James A Wells and Michael J Therien and William F DeGrado},

doi = {10.1021/jacs.4c18151},

issn = {1520-5126},

year  = {2025},

date = {2025-02-01},

urldate = {2025-02-01},

journal = {J Am Chem Soc},

abstract = { protein design provides a framework to test our understanding of protein function and build proteins with cofactors and functions not found in nature. Here, we report the design of proteins designed to bind powerful photooxidants and the evaluation of the use of these proteins to generate diffusible small-molecule reactive species. Because excited-state dynamics are influenced by the dynamics and hydration of a photooxidant's environment, it was important to not only design a binding site but also to evaluate its dynamic properties. Thus, we used computational design in conjunction with molecular dynamics (MD) simulations to design a protein, designated NBP (DI inding rotein), that held a naphthalenediimide (NDI), a powerful photooxidant, in a programmable molecular environment. Solution NMR confirmed the structure of the complex. We evaluated two NDI cofactors in this  protein using ultrafast pump-probe spectroscopy to evaluate light-triggered intra- and intermolecular electron transfer function. Moreover, we demonstrated the utility of this platform to activate multiple molecular probes for protein labeling.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38594044,

title = {Structural Survey of Antigen Recognition by Synthetic Human Antibodies},

author = {Maryna Gorelik and Shane Miersch and Sachdev S Sidhu},

doi = {10.1101/pdb.over107759},

issn = {1559-6095},

year  = {2025},

date = {2025-02-01},

urldate = {2025-02-01},

journal = {Cold Spring Harb Protoc},

volume = {2025},

number = {2},

pages = {pdb.over107759},

abstract = {Synthetic antibody libraries have been used extensively to isolate and optimize antibodies. To generate these libraries, the immunological diversity and the antibody framework(s) that supports it outside of the binding regions are carefully designed/chosen to ensure favorable functional and biophysical properties. In particular, minimalist, single-framework synthetic libraries pioneered by our group have yielded a vast trove of antibodies to a broad array of antigens. Here, we review their systematic and iterative development to provide insights into the design principles that make them a powerful tool for drug discovery. In addition, the ongoing accumulation of crystal structures of antigen-binding fragment (Fab)-antigen complexes generated with synthetic antibodies enables a deepening understanding of the structural determinants of antigen recognition and usage of immunoglobulin sequence diversity, which can assist in developing new strategies for antibody and library optimization. Toward this, we also survey here the structural landscape of a comprehensive and unbiased set of 50 distinct complexes derived from these libraries and compare it to a similar set of natural antibodies with the goal of better understanding how each achieves molecular recognition and whether opportunities exist for iterative improvement of synthetic libraries. From this survey, we conclude that despite the minimalist strategies used for design of these synthetic antibody libraries, the overall structural interaction landscapes are highly similar to natural repertoires. We also found, however, some key differences that can help guide the iterative design of new synthetic libraries via the introduction of positionally tailored diversity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39865354,

title = {Engineered protein G variants for multifunctional antibody-based assemblies},

author = {Tomasz Slezak and Kelly M O'Leary and Jinyang Li and Ahmed Rohaim and Elena K Davydova and Anthony A Kossiakoff},

doi = {10.1002/pro.70019},

issn = {1469-896X},

year  = {2025},

date = {2025-02-01},

urldate = {2025-02-01},

journal = {Protein Sci},

volume = {34},

number = {2},

pages = {e70019},

abstract = {We have developed a portfolio of antibody-based modules that can be prefabricated as standalone units and snapped together in plug-and-play fashion to create uniquely powerful multifunctional assemblies. The basic building blocks are derived from multiple pairs of native and modified Fab scaffolds and protein G (PG) variants engineered by phage display to introduce high pair-wise specificity. The variety of possible Fab-PG pairings provides a highly orthogonal system that can be exploited to perform challenging cell biology operations in a straightforward manner. The simplest manifestation allows multiplexed antigen detection using PG variants fused to fluorescently labeled SNAP-tags. Moreover, Fabs can be readily attached to a PG-Fc dimer module which acts as the core unit to produce plug-and-play IgG-like assemblies, and the utility can be further expanded to produce bispecific analogs using the "knobs into holes" strategy. These core PG-Fc dimer modules can be made and stored in bulk to produce off-the-shelf customized IgG entities in minutes, not days or weeks by just adding a Fab with the desired antigen specificity. In another application, the bispecific modalities form the building block for fabricating potent bispecific T-cell engagers (BiTEs), demonstrating their efficacy in cancer cell-killing assays. Additionally, the system can be adapted to include commercial antibodies as building blocks, greatly increasing the target space. Crystal structure analysis reveals that a few strategically positioned interactions engender the specificity between the Fab-PG variant pairs, requiring minimal changes to match the scaffolds for different possible combinations. This plug-and-play platform offers a user-friendly and versatile approach to enhance the functionality of antibody-based reagents in cell biology research.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39322765,

title = {Structural insights into translocation and tailored synthesis of hyaluronan},

author = {Ireneusz Górniak and Zachery Stephens and Satchal K Erramilli and Tomasz Gawda and Anthony A Kossiakoff and Jochen Zimmer},

doi = {10.1038/s41594-024-01389-1},

issn = {1545-9985},

year  = {2025},

date = {2025-01-01},

urldate = {2025-01-01},

journal = {Nat Struct Mol Biol},

volume = {32},

number = {1},

pages = {161--171},

abstract = {Hyaluronan (HA) is an essential component of the vertebrate extracellular matrix. It is a heteropolysaccharide of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) reaching several megadaltons in healthy tissues. HA is synthesized and translocated in a coupled reaction by HA synthase (HAS). Here, structural snapshots of HAS provide insights into HA biosynthesis, from substrate recognition to HA elongation and translocation. We monitor the extension of a GlcNAc primer with GlcA, reveal the coordination of the uridine diphosphate product by a conserved gating loop and capture the opening of a translocation channel to coordinate a translocating HA polymer. Furthermore, we identify channel-lining residues that modulate HA product lengths. Integrating structural and biochemical analyses suggests an avenue for polysaccharide engineering based on finely tuned enzymatic activity and HA coordination.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39792670,

title = {Ultrasensitive detection of intact SARS-CoV-2 particles in complex biofluids using microfluidic affinity capture},

author = {Daniel C Rabe and Adarsh Choudhury and Dasol Lee and Evelyn G Luciani and Uyen K Ho and Alex E Clark and Jeffrey E Glasgow and Sara Veiga and William A Michaud and Diane Capen and Elizabeth A Flynn and Nicola Hartmann and Aaron F Garretson and Alona Muzikansky and Marcia B Goldberg and Douglas S Kwon and Xu Yu and Aaron F Carlin and Yves Theriault and James A Wells and Jochen K Lennerz and Peggy S Lai and Sayed Ali Rabi and Anh N Hoang and Genevieve M Boland and Shannon L Stott},

doi = {10.1126/sciadv.adh1167},

issn = {2375-2548},

year  = {2025},

date = {2025-01-01},

urldate = {2025-01-01},

journal = {Sci Adv},

volume = {11},

number = {2},

pages = {eadh1167},

abstract = {Measuring virus in biofluids is complicated by confounding biomolecules coisolated with viral nucleic acids. To address this, we developed an affinity-based microfluidic device for specific capture of intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our approach used an engineered angiotensin-converting enzyme 2 to capture intact virus from plasma and other complex biofluids. Our device leverages a staggered herringbone pattern, nanoparticle surface coating, and processing conditions to achieve detection of as few as 3 viral copies per milliliter. We further validated our microfluidic assay on 103 plasma, 36 saliva, and 29 stool samples collected from unique patients with COVID-19, showing SARS-CoV-2 detection in 72% of plasma samples. Longitudinal monitoring in the plasma revealed our device's capacity for ultrasensitive detection of active viral infections over time. Our technology can be adapted to target other viruses using relevant cell entry molecules for affinity capture. This versatility underscores the potential for widespread application in viral load monitoring and disease management.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39793042,

title = {Mechanism-guided engineering of a minimal biological particle for genome editing},

author = {Wayne Ngo and Julia Peukes and Alisha Baldwin and Zhiwei Wayne Xue and Sidney Hwang and Robert R Stickels and Zhi Lin and Ansuman T Satpathy and James A Wells and Randy Schekman and Eva Nogales and Jennifer A Doudna},

doi = {10.1073/pnas.2413519121},

issn = {1091-6490},

year  = {2025},

date = {2025-01-01},

urldate = {2025-01-01},

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

volume = {122},

number = {1},

pages = {e2413519121},

abstract = {The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here, we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs and demonstrated increased editing in cell lines and therapeutically relevant primary human T cells. These results show that virally derived particles can be streamlined to create efficacious genome editing delivery vehicles with simpler production and manufacturing.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39627215,

title = {Conformational coupling between extracellular and transmembrane domains modulates holo-adhesion GPCR function},

author = {Szymon P Kordon and Kristina Cechova and Sumit J Bandekar and Katherine Leon and Przemysław Dutka and Gracie Siffer and Anthony A Kossiakoff and Reza Vafabakhsh and Demet Araç},

doi = {10.1038/s41467-024-54836-4},

issn = {2041-1723},

year  = {2024},

date = {2024-12-01},

urldate = {2024-12-01},

journal = {Nat Commun},

volume = {15},

number = {1},

pages = {10545},

abstract = {Adhesion G Protein-Coupled Receptors (aGPCRs) are key cell-adhesion molecules involved in numerous physiological functions. aGPCRs have large multi-domain extracellular regions (ECRs) containing a conserved GAIN domain that precedes their seven-pass transmembrane domain (7TM). Ligand binding and mechanical force applied on the ECR regulate receptor function. However, how the ECR communicates with the 7TM remains elusive, because the relative orientation and dynamics of the ECR and 7TM within a holoreceptor is unclear. Here, we describe the cryo-EM reconstruction of an aGPCR, Latrophilin3/ADGRL3, and reveal that the GAIN domain adopts a parallel orientation to the transmembrane region and has constrained movement. Single-molecule FRET experiments unveil three slow-exchanging FRET states of the ECR relative to the transmembrane region within the holoreceptor. GAIN-targeted antibodies, and cancer-associated mutations at the GAIN-7TM interface, alter FRET states, cryo-EM conformations, and receptor signaling. Altogether, this data demonstrates conformational and functional coupling between the ECR and 7TM, suggesting an ECR-mediated mechanism for aGPCR activation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39605704,

title = {CRISPRi-based screen of Autism Spectrum Disorder risk genes in microglia uncovers roles of  in microglia endocytosis and synaptic pruning},

author = {Olivia M Teter and Amanda McQuade and Venus Hagan and Weiwei Liang and Nina M Dräger and Sydney M Sattler and Brandon B Holmes and Vincent Cele Castillo and Vasileios Papakis and Kun Leng and Steven Boggess and Tomasz J Nowakowski and James Wells and Martin Kampmann},

doi = {10.1101/2024.06.01.596962},

issn = {2692-8205},

year  = {2024},

date = {2024-11-01},

urldate = {2024-11-01},

journal = {bioRxiv},

abstract = {Autism Spectrum Disorders (ASD) are a set of neurodevelopmental disorders with complex biology. The identification of ASD risk genes from exome-wide association studies and de novo variation analyses has enabled mechanistic investigations into how ASD-risk genes alter development. Most functional genomics studies have focused on the role of these genes in neurons and neural progenitor cells. However, roles for ASD risk genes in other cell types are largely uncharacterized. There is evidence from postmortem tissue that microglia, the resident immune cells of the brain, appear activated in ASD. Here, we used CRISPRi-based functional genomics to systematically assess the impact of ASD risk gene knockdown on microglia activation and phagocytosis. We developed an iPSC-derived microglia-neuron coculture system and high-throughput flow cytometry readout for synaptic pruning to enable parallel CRISPRi-based screening of phagocytosis of beads, synaptosomes, and synaptic pruning. Our screen identified , a high-confidence ASD risk genes, as a modifier of microglial synaptic pruning. We found that microglia with ADNP loss have altered endocytic trafficking, remodeled proteomes, and increased motility in coculture.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}