Publications

@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{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{pmid39383874,

title = {Cryo-EM structures of Clostridium perfringens enterotoxin bound to its human receptor, claudin-4},

author = {Sewwandi S Rathnayake and Satchal K Erramilli and Anthony A Kossiakoff and Alex J Vecchio},

doi = {10.1016/j.str.2024.09.015},

issn = {1878-4186},

year  = {2024},

date = {2024-11-01},

urldate = {2024-11-01},

journal = {Structure},

volume = {32},

number = {11},

pages = {1936--1951.e5},

abstract = {Clostridium perfringens enterotoxin (CpE) causes prevalent and deadly gastrointestinal disorders. CpE binds to receptors called claudins on the apical surfaces of small intestinal epithelium. Claudins normally regulate paracellular transport but are hijacked from doing so by CpE and are instead led to form claudin/CpE complexes. Claudin/CpE complexes are the building blocks of oligomeric β-barrel pores that penetrate the plasma membrane and induce gut cytotoxicity. Here, we present the structures of CpE in complex with its native claudin receptor in humans, claudin-4, using cryogenic electron microscopy. The structures reveal the architecture of the claudin/CpE complex, the residues used in binding, the orientation of CpE relative to the membrane, and CpE-induced changes to claudin-4. Further, structures and modeling allude to the biophysical procession from claudin/CpE complexes to cytotoxic β-barrel pores during pathogenesis. In full, this work proposes a model of claudin/CpE assembly and provides strategies to obstruct its formation to treat CpE diseases.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39605593,

title = {Biophysical Basis of Paracellular Barrier Modulation by a Pan-Claudin-Binding Molecule},

author = {Chinemerem P Ogbu and Alexandria M Mandriota and Xiangdong Liu and Mason de Las Alas and Srajan Kapoor and Jagrity Choudhury and Anthony A Kossiakoff and Michael E Duffey and Alex J Vecchio},

doi = {10.1101/2024.11.10.622873},

issn = {2692-8205},

year  = {2024},

date = {2024-11-01},

urldate = {2024-11-01},

journal = {bioRxiv},

abstract = {Claudins are a 27-member protein family that form and fortify specialized cell contacts in endothelium and epithelium called tight junctions. Tight junctions restrict paracellular transport across tissues by forming molecular barriers between cells. Claudin-binding molecules thus hold promise for modulating tight junction permeability to deliver drugs or as therapeutics to treat tight junction-linked disease. The development of claudin-binding molecules, however, is hindered by their intractability and small targetable surfaces. Here, we determine that a synthetic antibody fragment (sFab) we developed binds directly to 10 claudin subtypes with nanomolar affinity by targeting claudin's paracellular-exposed surface. Application of this sFab to cells that model intestinal epithelium show that it opens the paracellular barrier comparable to a known, but application limited, tight junction modulator. This novel pan-claudin-binding molecule can probe claudin or tight junction structure and holds potential as a broad modulator of tight junction permeability for basic or translational applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39651149,

title = {Molecular characterization of the archaic HLA-B*73:01 allele reveals presentation of a unique peptidome and skewed engagement by KIR2DL2},

author = {Philipp Ross and Hugo G Hilton and Jane Lodwick and Tomasz Slezak and Lisbeth A Guethlein and Curtis P McMurtrey and Alex S Han and Morten Nielsen and Daniel Yong and Charles L Dulberger and Kristof T Nolan and Sobhan Roy and Caitlin D Castro and William H Hildebrand and Minglei Zhao and Anthony Kossiakoff and Peter Parham and Erin J Adams},

doi = {10.1101/2024.11.25.625330},

issn = {2692-8205},

year  = {2024},

date = {2024-11-01},

urldate = {2024-11-01},

journal = {bioRxiv},

abstract = {HLA class I alleles of archaic origin may have been retained in modern humans because they provide immunity against diseases to which archaic humans had evolved resistance. According to this model, archaic introgressed alleles were somehow distinct from those that evolved in African populations. Here we show that HLA-B*73:01, a rare allotype with putative archaic origins, has a relatively rare peptide binding motif with an unusually long-tailed peptide length distribution. We also find that HLA-B*73:01 combines a restricted and unique peptidome with high-cell surface expression, characteristics that make it well-suited to combat one or a number of closely-related pathogens. Furthermore, a crystal structure of HLA-B*73:01 in complex with KIR2DL2 highlights differences from previously solved structures with HLA-C molecules. These molecular characteristics distinguish HLA-B*73:01 from other HLA class I alleles previously investigated and may have provided early modern human migrants that inherited this allele with a selective advantage as they colonized Europe and Asia.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37577460,

title = {Chaperone-assisted cryo-EM structure of  PhuR reveals molecular basis for heme binding},

author = {Paweł P Knejski and Satchal K Erramilli and Anthony A Kossiakoff},

doi = {10.1101/2023.08.01.551527},

issn = {2692-8205},

year  = {2024},

date = {2024-08-01},

urldate = {2024-08-01},

journal = {bioRxiv},

abstract = {Pathogenic bacteria, such as , depend on scavenging heme for the acquisition of iron, an essential nutrient. The TonB-dependent transporter (TBDT) PhuR is the major heme uptake protein in  clinical isolates. However, a comprehensive understanding of heme recognition and TBDT transport mechanisms, especially PhuR, remains limited. In this study, we employed single-particle cryogenic electron microscopy (cryo-EM) and a phage display-generated synthetic antibody (sAB) as a fiducial marker to enable the determination of a high-resolution (2.5 Å) structure of PhuR with a bound heme. Notably, the structure reveals iron coordination by Y529 on a conserved extracellular loop, shedding light on the role of tyrosine in heme binding. Biochemical assays and negative-stain EM demonstrated that the sAB specifically targets the heme-bound state of PhuR. These findings provide insights into PhuR's heme binding and offer a template for developing conformation-specific sABs against outer membrane proteins (OMPs) for structure-function investigations.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38233584,

title = {Covalent inhibition of pro-apoptotic BAX},

author = {Matthew W McHenry and Peiwen Shi and Christina M Camara and Daniel T Cohen and T Justin Rettenmaier and Utsarga Adhikary and Micah A Gygi and Ka Yang and Steven P Gygi and Thomas E Wales and John R Engen and James A Wells and Loren D Walensky},

doi = {10.1038/s41589-023-01537-6},

issn = {1552-4469},

year  = {2024},

date = {2024-08-01},

urldate = {2024-08-01},

journal = {Nat Chem Biol},

volume = {20},

number = {8},

pages = {1022--1032},

abstract = {BCL-2-associated X protein (BAX) is a promising therapeutic target for activating or restraining apoptosis in diseases of pathologic cell survival or cell death, respectively. In response to cellular stress, BAX transforms from a quiescent cytosolic monomer into a toxic oligomer that permeabilizes the mitochondria, releasing key apoptogenic factors. The mitochondrial lipid trans-2-hexadecenal (t-2-hex) sensitizes BAX activation by covalent derivatization of cysteine 126 (C126). In this study, we performed a disulfide tethering screen to discover C126-reactive molecules that modulate BAX activity. We identified covalent BAX inhibitor 1 (CBI1) as a compound that selectively derivatizes BAX at C126 and inhibits BAX activation by triggering ligands or point mutagenesis. Biochemical and structural analyses revealed that CBI1 can inhibit BAX by a dual mechanism of action: conformational constraint and competitive blockade of lipidation. These data inform a pharmacologic strategy for suppressing apoptosis in diseases of unwanted cell death by covalent targeting of BAX C126.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38308046,

title = {Antibody discovery identifies regulatory mechanisms of protein arginine deiminase 4},

author = {Xin Zhou and Sophie Kong and Allison Maker and Soumya G Remesh and Kevin K Leung and Kliment A Verba and James A Wells},

doi = {10.1038/s41589-023-01535-8},

issn = {1552-4469},

year  = {2024},

date = {2024-06-01},

urldate = {2024-06-01},

journal = {Nat Chem Biol},

volume = {20},

number = {6},

pages = {742--750},

abstract = {Unlocking the potential of protein arginine deiminase 4 (PAD4) as a drug target for rheumatoid arthritis requires a deeper understanding of its regulation. In this study, we use unbiased antibody selections to identify functional antibodies capable of either activating or inhibiting PAD4 activity. Through cryogenic-electron microscopy, we characterized the structures of these antibodies in complex with PAD4 and revealed insights into their mechanisms of action. Rather than steric occlusion of the substrate-binding catalytic pocket, the antibodies modulate PAD4 activity through interactions with allosteric binding sites adjacent to the catalytic pocket. These binding events lead to either alteration of the active site conformation or the enzyme oligomeric state, resulting in modulation of PAD4 activity. Our study uses antibody engineering to reveal new mechanisms for enzyme regulation and highlights the potential of using PAD4 agonist and antagonist antibodies for studying PAD4-dependency in disease models and future therapeutic development.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38683990,

title = {A general approach for selection of epitope-directed binders to proteins},

author = {Jie Zhou and Chau Q Le and Yun Zhang and James A Wells},

doi = {10.1073/pnas.2317307121},

issn = {1091-6490},

year  = {2024},

date = {2024-05-01},

urldate = {2024-05-01},

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

volume = {121},

number = {19},

pages = {e2317307121},

abstract = {Directing antibodies to a particular epitope among many possible on a target protein is a significant challenge. Here, we present a simple and general method for epitope-directed selection (EDS) using a differential phage selection strategy. This involves engineering the protein of interest (POI) with the epitope of interest (EOI) mutated using a systematic bioinformatics algorithm to guide the local design of an EOI decoy variant. Using several alternating rounds of negative selection with the EOI decoy variant followed by positive selection on the wild-type POI, we were able to identify highly specific and potent antibodies to five different EOI antigens that bind and functionally block known sites of proteolysis. Among these, we developed highly specific antibodies that target the proteolytic site on the CUB domain containing protein 1 (CDCP1) to prevent its proteolysis allowing us to study the cellular maturation of this event that triggers malignancy. We generated antibodies that recognize the junction between the pro- and catalytic domains for three different matrix metalloproteases (MMPs), MMP1, MMP3, and MMP9, that selectively block activation of each of these enzymes and impair cell migration. We targeted a proteolytic epitope on the cell surface receptor, EPH Receptor A2 (EphA2), that is known to transform it from a tumor suppressor to an oncoprotein. We believe that the EDS method greatly facilitates the generation of antibodies to specific EOIs on a wide range of proteins and enzymes for broad therapeutic and diagnostic applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38493204,

title = {Challenges in the discovery of tumor-specific alternative splicing-derived cell-surface antigens in glioma},

author = {Takahide Nejo and Lin Wang and Kevin K Leung and Albert Wang and Senthilnath Lakshmanachetty and Marco Gallus and Darwin W Kwok and Chibo Hong and Lee H Chen and Diego A Carrera and Michael Y Zhang and Nicholas O Stevers and Gabriella C Maldonado and Akane Yamamichi and Payal B Watchmaker and Akul Naik and Anny Shai and Joanna J Phillips and Susan M Chang and Arun P Wiita and James A Wells and Joseph F Costello and Aaron A Diaz and Hideho Okada},

doi = {10.1038/s41598-024-56684-0},

issn = {2045-2322},

year  = {2024},

date = {2024-03-01},

urldate = {2024-03-01},

journal = {Sci Rep},

volume = {14},

number = {1},

pages = {6362},

abstract = {Despite advancements in cancer immunotherapy, solid tumors remain formidable challenges. In glioma, profound inter- and intra-tumoral heterogeneity of antigen landscape hampers therapeutic development. Therefore, it is critical to consider alternative sources to expand the repertoire of targetable (neo-)antigens and improve therapeutic outcomes. Accumulating evidence suggests that tumor-specific alternative splicing (AS) could be an untapped reservoir of antigens. In this study, we investigated tumor-specific AS events in glioma, focusing on those predicted to generate major histocompatibility complex (MHC)-presentation-independent, cell-surface antigens that could be targeted by antibodies and chimeric antigen receptor-T cells. We systematically analyzed bulk RNA-sequencing datasets comparing 429 tumor samples (from The Cancer Genome Atlas) and 9166 normal tissue samples (from the Genotype-Tissue Expression project), and identified 13 AS events in 7 genes predicted to be expressed in more than 10% of the patients, including PTPRZ1 and BCAN, which were corroborated by an external RNA-sequencing dataset. Subsequently, we validated our predictions and elucidated the complexity of the isoforms using full-length transcript amplicon sequencing on patient-derived glioblastoma cells. However, analyses of the RNA-sequencing datasets of spatially mapped and longitudinally collected clinical tumor samples unveiled remarkable spatiotemporal heterogeneity of the candidate AS events. Furthermore, proteomics analysis did not reveal any peptide spectra matching the putative antigens. Our investigation illustrated the diverse characteristics of the tumor-specific AS events and the challenges of antigen exploration due to their notable spatiotemporal heterogeneity and elusive nature at the protein levels. Redirecting future efforts toward intracellular, MHC-presented antigens could offer a more viable avenue.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38448587,

title = {Oxidative cyclization reagents reveal tryptophan cation-π interactions},

author = {Xiao Xie and Patrick J Moon and Steven W M Crossley and Amanda J Bischoff and Dan He and Gen Li and Nam Dao and Angel Gonzalez-Valero and Audrey G Reeves and Jeffrey M McKenna and Susanna K Elledge and James A Wells and F Dean Toste and Christopher J Chang},

doi = {10.1038/s41586-024-07140-6},

issn = {1476-4687},

year  = {2024},

date = {2024-03-01},

urldate = {2024-03-01},

journal = {Nature},

volume = {627},

number = {8004},

pages = {680--687},

abstract = {Methods for selective covalent modification of amino acids on proteins can enable a diverse array of applications, spanning probes and modulators of protein function to proteomics. Owing to their high nucleophilicity, cysteine and lysine residues are the most common points of attachment for protein bioconjugation chemistry through acid-base reactivity. Here we report a redox-based strategy for bioconjugation of tryptophan, the rarest amino acid, using oxaziridine reagents that mimic oxidative cyclization reactions in indole-based alkaloid biosynthetic pathways to achieve highly efficient and specific tryptophan labelling. We establish the broad use of this method, termed tryptophan chemical ligation by cyclization (Trp-CLiC), for selectively appending payloads to tryptophan residues on peptides and proteins with reaction rates that rival traditional click reactions and enabling global profiling of hyper-reactive tryptophan sites across whole proteomes. Notably, these reagents reveal a systematic map of tryptophan residues that participate in cation-π interactions, including functional sites that can regulate protein-mediated phase-separation processes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38158383,

title = {The Molecular Basis of Human ALKBH3 Mediated RNA N -methyladenosine (m A) Demethylation},

author = {Lin Zhang and Hong-Chao Duan and Marcin Paduch and Jingyan Hu and Chi Zhang and Yajuan Mu and Houwen Lin and Chuan He and Anthony A Kossiakoff and Guifang Jia and Liang Zhang},

doi = {10.1002/anie.202313900},

issn = {1521-3773},

year  = {2024},

date = {2024-02-01},

urldate = {2024-02-01},

journal = {Angew Chem Int Ed Engl},

volume = {63},

number = {7},

pages = {e202313900},

abstract = {N -methyladenosine (m A) is a prevalent post-transcriptional RNA modification, and the distribution and dynamics of the modification play key epitranscriptomic roles in cell development. At present, the human AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family member ALKBH3 is the only known mRNA m A demethylase, but its catalytic mechanism remains unclear. Here, we present the structures of ALKBH3-oligo crosslinked complexes obtained with the assistance of a synthetic antibody crystallization chaperone. Structural and biochemical results showed that ALKBH3 utilized two β-hairpins (β4-loop-β5 and β'-loop-β'') and the α2 helix to facilitate single-stranded substrate binding. Moreover, a bubble-like region around Asp194 and a key residue inside the active pocket (Thr133) enabled specific recognition and demethylation of m A- and 3-methylcytidine (m C)-modified substrates. Mutation of Thr133 to the corresponding residue in the AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family members FTO or ALKBH5 converted ALKBH3 substrate selectivity from m A to N -methyladenosine (m A), as did Asp194 deletion. Our findings provide a molecular basis for understanding the mechanisms of substrate recognition and m A demethylation by ALKBH3. This study is expected to aid structure-guided design of chemical probes for further functional studies and therapeutic applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38062152,

title = {Extracellular targeted protein degradation: an emerging modality for drug discovery},

author = {James A Wells and Kaan Kumru},

doi = {10.1038/s41573-023-00833-z},

issn = {1474-1784},

year  = {2024},

date = {2024-02-01},

urldate = {2024-02-01},

journal = {Nat Rev Drug Discov},

volume = {23},

number = {2},

pages = {126--140},

abstract = {Targeted protein degradation (TPD) has emerged in the past decade as a major new drug modality to remove intracellular proteins with bispecific small molecules that recruit the protein of interest (POI) to an E3 ligase for degradation in the proteasome. Unlike classic occupancy-based drugs, intracellular TPD (iTPD) eliminates the target and works catalytically, and so can be more effective and sustained, with lower dose requirements. Recently, this approach has been expanded to the extracellular proteome, including both secreted and membrane proteins. Extracellular targeted protein degradation (eTPD) uses bispecific antibodies, conjugates or small molecules to degrade extracellular POIs by trafficking them to the lysosome for degradation. Here, we focus on recent advances in eTPD, covering degrader systems, targets, molecular designs and parameters to advance them. Now almost any protein, intracellular or extracellular, is addressable in principle with TPD.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38292613,

title = {Photoproximity Labeling from Single Catalyst Sites Allows Calibration and Increased Resolution for Carbene Labeling of Protein Partners In Vitro and on Cells},

author = {Thomas G Bartholow and Paul W W Burroughs and Susanna K Elledge and James R Byrnes and Lisa L Kirkemo and Virginia Garda and Kevin K Leung and James A Wells},

doi = {10.1021/acscentsci.3c01473},

issn = {2374-7943},

year  = {2024},

date = {2024-01-01},

urldate = {2024-01-01},

journal = {ACS Cent Sci},

volume = {10},

number = {1},

pages = {199--208},

abstract = {The cell surface proteome (surfaceome) plays a pivotal role in virtually all extracellular biology, and yet we are only beginning to understand the protein complexes formed in this crowded environment. Recently, a high-resolution approach (μMap) was described that utilizes multiple iridium-photocatalysts attached to a secondary antibody, directed to a primary antibody of a protein of interest, to identify proximal neighbors by light-activated conversion of a biotin-diazirine to a highly reactive carbene followed by LC/MS (Geri, J. B.; Oakley, J. V.; Reyes-Robles, T.; Wang, T.; McCarver, S. J.; White, C. H.; Rodriguez-Rivera, F. P.; Parker, D. L.; Hett, E. C.; Fadeyi, O. O.; Oslund, R. C.; MacMillan, D. W. C. , , 1091-1097). Here we calibrated the spatial resolution for carbene labeling using site-specific conjugation of a single photocatalyst to a primary antibody drug, trastuzumab (Traz), in complex with its structurally well-characterized oncogene target, HER2. We observed relatively uniform carbene labeling across all amino acids, and a maximum distance of ∼110 Å from the fixed photocatalyst. When targeting HER2 overexpression cells, we identified 20 highly enriched HER2 neighbors, compared to a nonspecific membrane tethered catalyst. These studies identify new HER2 interactors and calibrate the radius of carbene photoprobe labeling for the surfaceome.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37400538,

title = {Discovery of VH domains that allosterically inhibit ENPP1},

author = {Paige E Solomon and Colton J Bracken and Jacqueline A Carozza and Haoqing Wang and Elizabeth P Young and Alon Wellner and Chang C Liu and E Alejandro Sweet-Cordero and Lingyin Li and James A Wells},

doi = {10.1038/s41589-023-01368-5},

issn = {1552-4469},

year  = {2024},

date = {2024-01-01},

urldate = {2024-01-01},

journal = {Nat Chem Biol},

volume = {20},

number = {1},

pages = {30--41},

abstract = {Ectodomain phosphatase/phosphodiesterase-1 (ENPP1) is overexpressed on cancer cells and functions as an innate immune checkpoint by hydrolyzing extracellular cyclic guanosine monophosphate adenosine monophosphate (cGAMP). Biologic inhibitors have not yet been reported and could have substantial therapeutic advantages over current small molecules because they can be recombinantly engineered into multifunctional formats and immunotherapies. Here we used phage and yeast display coupled with in cellulo evolution to generate variable heavy (VH) single-domain antibodies against ENPP1 and discovered a VH domain that allosterically inhibited the hydrolysis of cGAMP and adenosine triphosphate (ATP). We solved a 3.2 Å-resolution cryo-electron microscopy structure for the VH inhibitor complexed with ENPP1 that confirmed its new allosteric binding pose. Finally, we engineered the VH domain into multispecific formats and immunotherapies, including a bispecific fusion with an anti-PD-L1 checkpoint inhibitor that showed potent cellular activity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37961561,

title = {Multi-scale photocatalytic proximity labeling reveals cell surface neighbors on and between cells},

author = {Zhi Lin and Kaitlin Schaefer and Irene Lui and Zi Yao and Andrea Fossati and Danielle L Swaney and Ajikarunia Palar and Andrej Sali and James A Wells},

doi = {10.1101/2023.10.28.564055},

issn = {2692-8205},

year  = {2023},

date = {2023-10-01},

urldate = {2023-10-01},

journal = {bioRxiv},

abstract = {The cell membrane proteome is the primary biohub for cell communication, yet we are only beginning to understand the dynamic protein neighborhoods that form on the cell surface and between cells. Proximity labeling proteomics (PLP) strategies using chemically reactive probes are powerful approaches to yield snapshots of protein neighborhoods but are currently limited to one single resolution based on the probe labeling radius. Here, we describe a multi-scale PLP method with tunable resolution using a commercially available histological dye, Eosin Y, which upon visible light illumination, activates three different photo-probes with labeling radii ranging from ∼100 to 3000 Å. We applied this platform to profile neighborhoods of the oncogenic epidermal growth factor receptor (EGFR) and orthogonally validated >20 neighbors using immuno-assays and AlphaFold-Multimer prediction that generated plausible binary interaction models. We further profiled the protein neighborhoods of cell-cell synapses induced by bi-specific T-cell engagers (BiTEs) and chimeric antigen receptor (CAR)T cells at longer length scales. This integrated multi-scale PLP platform maps local and distal protein networks on cell surfaces and between cells. We believe this information will aid in the systematic construction of the cell surface interactome and reveal new opportunities for immunotherapeutics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37394032,

title = {Conformation-specific Synthetic Antibodies Discriminate Multiple Functional States of the Ion Channel CorA},

author = {Satchal K Erramilli and Pawel K Dominik and Dawid Deneka and Piotr Tokarz and Sangwoo S Kim and Bharat G Reddy and Blazej M Skrobek and Olivier Dalmas and Eduardo Perozo and Anthony A Kossiakoff},

doi = {10.1016/j.jmb.2023.168192},

issn = {1089-8638},

year  = {2023},

date = {2023-09-01},

urldate = {2023-09-01},

journal = {J Mol Biol},

volume = {435},

number = {17},

pages = {168192},

abstract = {CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg, and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg. Two sABs from these selections, C12 and C18, showed different degrees of Mg-sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg-depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg-depleted conditions. We used X-ray crystallography to determine a structure at 2.0 Å resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37723174,

title = {Structure of human drug transporters OATP1B1 and OATP1B3},

author = {Anca-Denise Ciută and Kamil Nosol and Julia Kowal and Somnath Mukherjee and Ana S Ramírez and Bruno Stieger and Anthony A Kossiakoff and Kaspar P Locher},

doi = {10.1038/s41467-023-41552-8},

issn = {2041-1723},

year  = {2023},

date = {2023-09-01},

urldate = {2023-09-01},

journal = {Nat Commun},

volume = {14},

number = {1},

pages = {5774},

abstract = {The organic anion transporting polypeptides OATP1B1 and OATP1B3 are membrane proteins that mediate uptake of drugs into the liver for subsequent conjugation and biliary excretion, a key step in drug elimination from the human body. Polymorphic variants of these transporters can cause reduced drug clearance and adverse drug effects such as statin-induced rhabdomyolysis, and co-administration of OATP substrates can lead to damaging drug-drug interaction. Despite their clinical relevance in drug disposition and pharmacokinetics, the structure and mechanism of OATPs are unknown. Here we present cryo-EM structures of human OATP1B1 and OATP1B3 bound to synthetic Fab fragments and in functionally distinct states. A single estrone-3-sulfate molecule is bound in a pocket located in the C-terminal half of OATP1B1. The shape and chemical nature of the pocket rationalize the preference for diverse organic anions and allow in silico docking of statins. The structure of OATP1B3 is determined in a drug-free state but reveals a bicarbonate molecule bound to the conserved signature motif and a histidine residue that is prevalent in OATPs exhibiting pH-dependent activity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}