Publications

@article{Bruce2024,

title = {Antigen‐binding fragments with improved crystal lattice packing and enhanced conformational flexibility at the elbow region as crystallization chaperones},

author = {Heather A. Bruce and Alexander U. Singer and Levi L. Blazer and Khanh Luu and Lynda Ploder and Alevtina Pavlenco and Igor Kurinov and Jarrett J. Adams and Sachdev S. Sidhu},

doi = {10.1002/pro.5081},

issn = {1469-896X},

year  = {2024},

date = {2024-07-00},

urldate = {2024-07-00},

journal = {Protein Science},

volume = {33},

number = {7},

publisher = {Wiley},

abstract = {<jats:title>Abstract</jats:title><jats:p>It has been shown previously that a set of three modifications—termed S1, Crystal Kappa, and elbow—act synergistically to improve the crystallizability of an antigen‐binding fragment (Fab) framework. Here, we prepared a phage‐displayed library and performed crystallization screenings to identify additional substitutions—located near the heavy‐chain elbow region—which cooperate with the S1, Crystal Kappa, and elbow modifications to increase expression and improve crystallizability of the Fab framework even further. One substitution (K141Q) supports the signature Crystal Kappa‐mediated Fab:Fab crystal lattice packing interaction. Another substitution (E172G) improves the compatibility of the elbow modification with the Fab framework by alleviating some of the strain incurred by the shortened and bulkier elbow linker region. A third substitution (F170W) generates a split‐Fab conformation, resulting in a powerful crystal lattice packing interaction comprising the biological interaction interface between the variable heavy and light chain domains. In sum, we have used K141Q, E172G, and F170W substitutions—which complement the S1, Crystal Kappa, and elbow modifications—to generate a set of highly crystallizable Fab frameworks that can be used as chaperones to enable facile elucidation of Fab:antigen complex structures by x‐ray crystallography.</jats:p>},

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{pmid38843544,

title = {Development of Luminescent Biosensors for Calprotectin},

author = {Tong Lan and Tomasz Slezak and Jinyue Pu and Julia Zinkus-Boltz and Sarbani Adhikari and Joel R Pekow and Vibha Taneja and Joaquin Zuniga and Itzel A Gómez-García and Nora Regino-Zamarripa and Mushtaq Ahmed and Shabaana A Khader and David T Rubin and Anthony A Kossiakoff and Bryan C Dickinson},

doi = {10.1021/acschembio.4c00265},

issn = {1554-8937},

year  = {2024},

date = {2024-06-01},

urldate = {2024-06-01},

journal = {ACS Chem Biol},

volume = {19},

number = {6},

pages = {1250--1259},

abstract = {Calprotectin, a metal ion-binding protein complex, plays a crucial role in the innate immune system and has gained prominence as a biomarker for various intestinal and systemic inflammatory and infectious diseases, including inflammatory bowel disease (IBD) and tuberculosis (TB). Current clinical testing methods rely on enzyme-linked immunosorbent assays (ELISAs), limiting accessibility and convenience. In this study, we introduce the Fab-Enabled Split-luciferase Calprotectin Assay (FESCA), a novel quantitative method for calprotectin measurement. FESCA utilizes two new fragment antigen binding proteins (Fabs), CP16 and CP17, that bind to different epitopes of the calprotectin complex. These Fabs are fused with split NanoLuc luciferase fragments, enabling the reconstitution of active luciferase upon binding to calprotectin either in solution or in varied immobilized assay formats. FESCA's output luminescence can be measured with standard laboratory equipment as well as consumer-grade cell phone cameras. FESCA can detect physiologically relevant calprotectin levels across various sample types, including serum, plasma, and whole blood. Notably, FESCA can detect abnormally elevated native calprotectin from TB patients. In summary, FESCA presents a convenient, low-cost, and quantitative method for assessing calprotectin levels in various biological samples, with the potential to improve the diagnosis and monitoring of inflammatory diseases, especially in at-home or point-of-care settings.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38979142,

title = {Structural Insights into the Roles of PARP4 and NAD  in the Human Vault Cage},

author = {Jane E Lodwick and Rong Shen and Satchal Erramilli and Yuan Xie and Karolina Roganowicz and Anthony A Kossiakoff and Minglei Zhao},

doi = {10.1101/2024.06.27.601040},

issn = {2692-8205},

year  = {2024},

date = {2024-06-01},

urldate = {2024-06-01},

journal = {bioRxiv},

abstract = {Vault is a massive ribonucleoprotein complex found across Eukaryota. The major vault protein (MVP) oligomerizes into an ovular cage, which contains several minor vault components (MVCs) and is thought to transport transiently bound "cargo" molecules. Vertebrate vaults house a poly (ADP-ribose) polymerase (known as PARP4 in humans), which is the only MVC with known enzymatic activity. Despite being discovered decades ago, the molecular basis for PARP4's interaction with MVP remains unclear. In this study, we determined the structure of the human vault cage in complex with PARP4 and its enzymatic substrate NAD  . The structures reveal atomic-level details of the protein-binding interface, as well as unexpected NAD  -binding pockets within the interior of the vault cage. In addition, proteomics data show that human vaults purified from wild-type and PARP4-depleted cells interact with distinct subsets of proteins. Our results thereby support a model in which PARP4's specific incorporation into the vault cage helps to regulate vault's selection of cargo and its subcellular localization. Further, PARP4's proximity to MVP's NAD  -binding sites could support its enzymatic function within the vault.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38886509,

title = {Structural and biophysical insights into targeting of claudin-4 by a synthetic antibody fragment},

author = {Satchal K Erramilli and Pawel K Dominik and Chinemerem P Ogbu and Anthony A Kossiakoff and Alex J Vecchio},

doi = {10.1038/s42003-024-06437-6},

issn = {2399-3642},

year  = {2024},

date = {2024-06-01},

urldate = {2024-06-01},

journal = {Commun Biol},

volume = {7},

number = {1},

pages = {733},

abstract = {Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties-these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes using cryogenic electron microscopy. Structural and biophysical results reveal this sFabs mechanism of select binding to human claudin-4 over other homologous claudins and establish the ability of sFabs to bind hard-to-target claudins to probe tight junction structure and function. The findings provide a framework for tight junction modulation by sFabs for tissue-selective therapies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38693257,

title = {Structural and molecular basis of choline uptake into the brain by FLVCR2},

author = {Rosemary J Cater and Dibyanti Mukherjee and Eva Gil-Iturbe and Satchal K Erramilli and Ting Chen and Katie Koo and Nicolás Santander and Andrew Reckers and Brian Kloss and Tomasz Gawda and Brendon C Choy and Zhening Zhang and Aditya Katewa and Amara Larpthaveesarp and Eric J Huang and Scott W J Mooney and Oliver B Clarke and Sook Wah Yee and Kathleen M Giacomini and Anthony A Kossiakoff and Matthias Quick and Thomas Arnold and Filippo Mancia},

doi = {10.1038/s41586-024-07326-y},

issn = {1476-4687},

year  = {2024},

date = {2024-05-01},

urldate = {2024-05-01},

journal = {Nature},

volume = {629},

number = {8012},

pages = {704--709},

abstract = {Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.},

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{pmid37295821,

title = {Generation and Selection of Synthetic Human Antibody Libraries via Phage Display},

author = {Gianluca Veggiani and Sachdev S Sidhu},

doi = {10.1101/pdb.prot108347},

issn = {1559-6095},

year  = {2024},

date = {2024-04-01},

urldate = {2024-04-01},

journal = {Cold Spring Harb Protoc},

volume = {2024},

number = {4},

pages = {108347},

abstract = {Synthetic antibody libraries enable the development of antibodies that can recognize virtually any antigen, with affinity and specificity profiles that are superior to those of natural antibodies. By using highly stable and optimized frameworks, synthetic antibody libraries can be rapidly generated by precisely designing synthetic DNA, allowing absolute control over the position and chemical diversity introduced while expanding the sequence space for antigen recognition. Here, we describe a detailed protocol for the generation of highly diverse synthetic antibody phage display libraries based on a single framework, with diversity genetically incorporated by using finely designed mutagenic oligonucleotides. This general method enables the facile construction of large antibody libraries with precisely tunable features, resulting in the rapid development of recombinant antibodies for virtually any antigen.},

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{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{pmid38509088,

title = {Structure of antiviral drug bulevirtide bound to hepatitis B and D virus receptor protein NTCP},

author = {Hongtao Liu and Dariusz Zakrzewicz and Kamil Nosol and Rossitza N Irobalieva and Somnath Mukherjee and Rose Bang-Sørensen and Nora Goldmann and Sebastian Kunz and Lorenzo Rossi and Anthony A Kossiakoff and Stephan Urban and Dieter Glebe and Joachim Geyer and Kaspar P Locher},

doi = {10.1038/s41467-024-46706-w},

issn = {2041-1723},

year  = {2024},

date = {2024-03-01},

urldate = {2024-03-01},

journal = {Nat Commun},

volume = {15},

number = {1},

pages = {2476},

abstract = {Cellular entry of the hepatitis B and D viruses (HBV/HDV) requires binding of the viral surface polypeptide preS1 to the hepatobiliary transporter Na-taurocholate co-transporting polypeptide (NTCP). This interaction can be blocked by bulevirtide (BLV, formerly Myrcludex B), a preS1 derivative and approved drug for treating HDV infection. Here, to elucidate the basis of this inhibitory function, we determined a cryo-EM structure of BLV-bound human NTCP. BLV forms two domains, a plug lodged in the bile salt transport tunnel of NTCP and a string that covers the receptor's extracellular surface. The N-terminally attached myristoyl group of BLV interacts with the lipid-exposed surface of NTCP. Our structure reveals how BLV inhibits bile salt transport, rationalizes NTCP mutations that decrease the risk of HBV/HDV infection, and provides a basis for understanding the host specificity of HBV/HDV. Our results provide opportunities for structure-guided development of inhibitors that target HBV/HDV docking to NTCP.},

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{pmid38464178,

title = {Structural analysis and conformational dynamics of a holo-adhesion GPCR reveal interplay between extracellular and transmembrane domains},

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 = {10.1101/2024.02.25.581807},

issn = {2692-8205},

year  = {2024},

date = {2024-02-01},

urldate = {2024-02-01},

journal = {bioRxiv},

abstract = {Adhesion G Protein-Coupled Receptors (aGPCRs) are key cell-adhesion molecules involved in numerous physiological functions. aGPCRs have large multi-domain extracellular regions (ECR) 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 membrane and has constrained movement. Single-molecule FRET experiments unveil three slow-exchanging FRET states of the ECR relative to the 7TM 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 of aGPCR activation.},

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{pmid37945533,

title = {Engineered antigen-binding fragments for enhanced crystallization of antibody:antigen complexes},

author = {Heather A Bruce and Alexander U Singer and Ekaterina V Filippova and Levi L Blazer and Jarrett J Adams and Leonie Enderle and Moshe Ben-David and Elizabeth H Radley and Daniel Y L Mao and Victor Pau and Stephen Orlicky and Frank Sicheri and Igor Kurinov and Shane Atwell and Anthony A Kossiakoff and Sachdev S Sidhu},

doi = {10.1002/pro.4824},

issn = {1469-896X},

year  = {2024},

date = {2024-01-01},

urldate = {2024-01-01},

journal = {Protein Sci},

volume = {33},

number = {1},

pages = {e4824},

abstract = {The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework. In this investigation, we applied the surface entropy reduction strategy coupled with phage-display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallize. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in an extraordinary improvement in Fab and Fab:antigen complex crystallizability, revealing a strong synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallization screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallization and how they complement each other in the process.},

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{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}

}