Publications: 2025

@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 = {2025-12-00},

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{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{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{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{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{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{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{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{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{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}

}