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{pmid39322765,

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

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

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

issn = {1545-9985},

year  = {2025},

date = {2025-01-01},

urldate = {2025-01-01},

journal = {Nat Struct Mol Biol},

volume = {32},

number = {1},

pages = {161--171},

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

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{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{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{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{pmid39345558,

title = {Structural insights into terminal arabinosylation biosynthesis of the mycobacterial cell wall arabinan},

author = {Yaqi Liu and Chelsea M Brown and Satchal Erramilli and Yi-Chia Su and Po-Sen Tseng and Yu-Jen Wang and Nam Ha Duong and Piotr Tokarz and Brian Kloss and Cheng-Ruei Han and Hung-Yu Chen and Jose Rodrigues and Margarida Archer and Todd L Lowary and Anthony A Kossiakoff and Phillip J Stansfeld and Rie Nygaard and Filippo Mancia},

doi = {10.1101/2024.09.17.613533},

issn = {2692-8205},

year  = {2024},

date = {2024-09-01},

urldate = {2024-09-01},

journal = {bioRxiv},

abstract = {The emergence of drug-resistant strains exacerbates the global challenge of tuberculosis caused by Mycobacterium tuberculosis (Mtb). Central to the pathogenicity of Mtb is its complex cell envelope, which serves as a barrier against both immune system and pharmacological attacks. Two key components of this envelope, arabinogalactan (AG) and lipoarabinomannan (LAM) are complex polysaccharides that contain integral arabinan domains important for cell wall structural and functional integrity. The arabinofuranosyltransferase AftB terminates the synthesis of these arabinan domains by catalyzing the addition of the addition of β-(1→2)-linked terminal arabinofuranose residues. Here, we present the cryo-EM structures of Mycobacterium chubuense AftB in its apo and donor substrate analog-bound form, determined to 2.9 Å and 3.4 Å resolution, respectively. Our structures reveal that AftB has a GT-C fold transmembrane (TM) domain comprised of eleven TM helices and a periplasmic cap domain. AftB has an irregular tube-shaped cavity that bridges the two proposed substrate binding sites. By integrating structural analysis, biochemical assays, and molecular dynamics simulations, we elucidate the molecular basis of the reaction mechanism of AftB and propose a model for catalysis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39345506,

title = {Mechanistic studies of mycobacterial glycolipid biosynthesis by the mannosyltransferase PimE},

author = {Yaqi Liu and Chelsea M Brown and Nuno Borges and Rodrigo N Nobre and Satchal Erramilli and Meagan Belcher Dufrisne and Brian Kloss and Sabrina Giacometti and Ana M Esteves and Cristina G Timoteo and Piotr Tokarz and Rosemary Cater and Yasu S Morita and Anthony A Kossiakoff and Helena Santos and Phillip J Stansfeld and Rie Nygaard and Filippo Mancia},

doi = {10.1101/2024.09.17.613550},

issn = {2692-8205},

year  = {2024},

date = {2024-09-01},

journal = {bioRxiv},

abstract = {Tuberculosis (TB), exceeded in mortality only by COVID-19 among global infectious diseases, is caused by Mycobacterium tuberculosis (Mtb). The pathogenicity of Mtb is largely attributed to its complex cell envelope, which includes a class of glycolipids called phosphatidyl-myo-inositol mannosides (PIMs), found uniquely in mycobacteria and its related corynebacterineae. These glycolipids maintain the integrity of the mycobacterial cell envelope, regulate its permeability, and mediate host-pathogen interactions. PIMs consist of a phosphatidyl-myo-inositol core decorated with one to six mannose residues and up to four acyl chains. The mannosyltransferase PimE catalyzes the transfer of the fifth PIM mannose residue from a polyprenyl phosphate-mannose (PPM) donor. This step in the biosynthesis of higher-order PIMs contributes to the proper assembly and function of the mycobacterial cell envelope; however, the structural basis for substrate recognition and the catalytic mechanism of PimE remain poorly understood. Here, we present the cryo-electron microscopy (cryo-EM) structures of PimE from Mycobacterium abscessus captured in its apo form and in a product-bound complex with the reaction product Ac1PIM5 and the by-product polyprenyl phosphate (PP), determined at 3.0 Å and 3.5 Å, respectively. The structures reveal the active site within a distinctive binding cavity that accommodates both donor and acceptor substrates/products. Within the cavity, we identified residues involved in substrate coordination and catalysis, which we confirmed through in vitro enzymatic assays and further validated by in vivo complementation experiments. Molecular dynamics simulations were applied to identify the access pathways and the dynamics involved in substrate binding. Integrating structural, biochemical, genetic, and computational experiments, our study provides comprehensive insights into how PimE functions, opening potential avenues for development of novel anti-TB therapeutics.},

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},

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{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},

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},

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{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},

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{pmid37873173,

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 Grez and Andrew Reckers and Brian Kloss and Tomasz Gawda and Brendon C Choy and Zhening Zheng and Oliver B Clarke and Sook Wah Yee and Anthony A Kossiakoff and Matthias Quick and Thomas Arnold and Filippo Mancia},

doi = {10.1101/2023.10.05.561059},

issn = {2692-8205},

year  = {2023},

date = {2023-10-01},

journal = {bioRxiv},

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 has eluded the field for over fifty years. The MFS transporter FLVCR1 was recently determined to be a choline transporter, and while this protein is not highly expressed at the blood-brain barrier (BBB), its relative FLVCR2 is. Previous studies have shown that mutations in human  cause cerebral vascular abnormalities, hydrocephalus, and embryonic lethality, but the physiological role of FLVCR2 is unknown. Here, we demonstrate both  and  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 the inward- and outward-facing states using cryo-electron microscopy to 2.49 and 2.77 Å resolution, respectively. 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 neurotherapeutics into the brain.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37474505,

title = {Antiviral HIV-1 SERINC restriction factors disrupt virus membrane asymmetry},

author = {Susan A Leonhardt and Michael D Purdy and Jonathan R Grover and Ziwei Yang and Sandra Poulos and William E McIntire and Elizabeth A Tatham and Satchal K Erramilli and Kamil Nosol and Kin Kui Lai and Shilei Ding and Maolin Lu and Pradeep D Uchil and Andrés Finzi and Alan Rein and Anthony A Kossiakoff and Walther Mothes and Mark Yeager},

doi = {10.1038/s41467-023-39262-2},

issn = {2041-1723},

year  = {2023},

date = {2023-07-01},

journal = {Nat Commun},

volume = {14},

number = {1},

pages = {4368},

abstract = {The host proteins SERINC3 and SERINC5 are HIV-1 restriction factors that reduce infectivity when incorporated into the viral envelope. The HIV-1 accessory protein Nef abrogates incorporation of SERINCs via binding to intracellular loop 4 (ICL4). Here, we determine cryoEM maps of full-length human SERINC3 and an ICL4 deletion construct, which reveal that hSERINC3 is comprised of two α-helical bundles connected by a ~ 40-residue, highly tilted, "crossmember" helix. The design resembles non-ATP-dependent lipid transporters. Consistently, purified hSERINCs reconstituted into proteoliposomes induce flipping of phosphatidylserine (PS), phosphatidylethanolamine and phosphatidylcholine. Furthermore, SERINC3, SERINC5 and the scramblase TMEM16F expose PS on the surface of HIV-1 and reduce infectivity, with similar results in MLV. SERINC effects in HIV-1 and MLV are counteracted by Nef and GlycoGag, respectively. Our results demonstrate that SERINCs are membrane transporters that flip lipids, resulting in a loss of membrane asymmetry that is strongly correlated with changes in Env conformation and loss of infectivity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid37502840,

title = {ACKR3-arrestin2/3 complexes reveal molecular consequences of GRK-dependent barcoding},

author = {Qiuyan Chen and Christopher T Schafer and Somnath Mukherjee and Martin Gustavsson and Parth Agrawal and Xin-Qiu Yao and Anthony A Kossiakoff and Tracy M Handel and John J G Tesmer},

doi = {10.1101/2023.07.18.549504},

issn = {2692-8205},

year  = {2023},

date = {2023-07-01},

journal = {bioRxiv},

abstract = {Atypical chemokine receptor 3 (ACKR3, also known as CXCR7) is a scavenger receptor that regulates extracellular levels of the chemokine CXCL12 to maintain responsiveness of its partner, the G protein-coupled receptor (GPCR), CXCR4. ACKR3 is notable because it does not couple to G proteins and instead is completely biased towards arrestins. Our previous studies revealed that GRK2 and GRK5 install distinct distributions of phosphates (or "barcodes") on the ACKR3 carboxy terminal tail, but how these unique barcodes drive different cellular outcomes is not understood. It is also not known if arrestin2 (Arr2) and 3 (Arr3) bind to these barcodes in distinct ways. Here we report cryo-electron microscopy structures of Arr2 and Arr3 in complex with ACKR3 phosphorylated by either GRK2 or GRK5. Unexpectedly, the finger loops of Arr2 and 3 directly insert into the detergent/membrane instead of the transmembrane core of ACKR3, in contrast to previously reported "core" GPCR-arrestin complexes. The distance between the phosphorylation barcode and the receptor transmembrane core regulates the interaction mode of arrestin, alternating between a tighter complex for GRK5 sites and heterogenous primarily "tail only" complexes for GRK2 sites. Arr2 and 3 bind at different angles relative to the core of ACKR3, likely due to differences in membrane/micelle anchoring at their C-edge loops. Our structural investigations were facilitated by Fab7, a novel Fab that binds both Arr2 and 3 in their activated states irrespective of receptor or phosphorylation status, rendering it a potentially useful tool to aid structure determination of any native GPCR-arrestin complex. The structures provide unprecedented insight into how different phosphorylation barcodes and arrestin isoforms can globally affect the configuration of receptor-arrestin complexes. These differences may promote unique downstream intracellular interactions and cellular responses. Our structures also suggest that the 100% bias of ACKR3 for arrestins is driven by the ability of arrestins, but not G proteins, to bind GRK-phosphorylated ACKR3 even when excluded from the receptor cytoplasmic binding pocket.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid36928458,

title = {Structural basis for assembly and lipid-mediated gating of LRRC8A:C volume-regulated anion channels},

author = {David M Kern and Julia Bleier and Somnath Mukherjee and Jennifer M Hill and Anthony A Kossiakoff and Ehud Y Isacoff and Stephen G Brohawn},

doi = {10.1038/s41594-023-00944-6},

issn = {1545-9985},

year  = {2023},

date = {2023-06-01},

journal = {Nat Struct Mol Biol},

volume = {30},

number = {6},

pages = {841--852},

abstract = {Leucine-rich repeat-containing protein 8 (LRRC8) family members form volume-regulated anion channels activated by hypoosmotic cell swelling. LRRC8 channels are ubiquitously expressed in vertebrate cells as heteromeric assemblies of LRRC8A (SWELL1) and LRRC8B-E subunits. Channels of different subunit composition have distinct properties that explain the functional diversity of LRRC8 currents across cell types. However, the basis for heteromeric LRRC8 channel assembly and function is unknown. Here we leverage a fiducial-tagging strategy to determine single-particle cryo-EM structures of heterohexameric LRRC8A:C channels in multiple conformations. Compared to homomers, LRRC8A:C channels show pronounced differences in architecture due to heterotypic LRR interactions that displace subunits away from the conduction axis and poise the channel for activation. Structures and functional studies further reveal that lipids embedded in the channel pore block ion conduction in the closed state. These results provide insight into determinants for heteromeric LRRC8 channel assembly, activity and gating by lipids.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid36977410,

title = {Characterization of synthetic antigen binding fragments targeting Toc75 for the isolation of TOC in A. thaliana and P. sativum},

author = {Karthik Srinivasan and Satchal K Erramilli and Srinivas Chakravarthy and Adrian Gonzalez and Anthony Kossiakoff and Nicholas Noinaj},

doi = {10.1016/j.str.2023.03.002},

issn = {1878-4186},

year  = {2023},

date = {2023-05-01},

journal = {Structure},

volume = {31},

number = {5},

pages = {595--606.e5},

abstract = {Roughly 95% of the proteins that make up the chloroplast must be imported from the cytoplasm. The machinery responsible for the translocation of these cargo proteins is called the translocon at the outer membrane of chloroplast (TOC). The TOC core consists of three proteins, Toc34, Toc75, and Toc159; no high-resolution structure has been solved of fully assembled TOC from plants. Efforts toward determining the structure of the TOC have been hindered almost entirely by difficulties in producing sufficient yields for structural studies. In this study, we introduce an innovative method that utilizes synthetic antigen binding fragments (sABs) to isolate TOC directly from wild-type plant biomass including A. thaliana and P. sativum. Binding between the sABs and the POTRA domains was characterized by size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS), X-ray crystallography, and isothermal titration calorimetry. We also demonstrate the isolation of the TOC from P. sativum, laying the framework for large-scale isolation and purification of TOC for functional and structural studies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid36604564,

title = {Structure, sequon recognition and mechanism of tryptophan C-mannosyltransferase},

author = {Joël S Bloch and Alan John and Runyu Mao and Somnath Mukherjee and Jérémy Boilevin and Rossitza N Irobalieva and Tamis Darbre and Nichollas E Scott and Jean-Louis Reymond and Anthony A Kossiakoff and Ethan D Goddard-Borger and Kaspar P Locher},

doi = {10.1038/s41589-022-01219-9},

issn = {1552-4469},

year  = {2023},

date = {2023-05-01},

journal = {Nat Chem Biol},

volume = {19},

number = {5},

pages = {575--584},

abstract = {C-linked glycosylation is essential for the trafficking, folding and function of secretory and transmembrane proteins involved in cellular communication processes. The tryptophan C-mannosyltransferase (CMT) enzymes that install the modification attach a mannose to the first tryptophan of WxxW/C sequons in nascent polypeptide chains by an unknown mechanism. Here, we report cryogenic-electron microscopy structures of Caenorhabditis elegans CMT in four key states: apo, acceptor peptide-bound, donor-substrate analog-bound and as a trapped ternary complex with both peptide and a donor-substrate mimic bound. The structures indicate how the C-mannosylation sequon is recognized by this CMT and its paralogs, and how sequon binding triggers conformational activation of the donor substrate: a process relevant to all glycosyltransferase C superfamily enzymes. Our structural data further indicate that the CMTs adopt an unprecedented electrophilic aromatic substitution mechanism to enable the C-glycosylation of proteins. These results afford opportunities for understanding human disease and therapeutic targeting of specific CMT paralogs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid36746957,

title = {Isoform- and ligand-specific modulation of the adhesion GPCR ADGRL3/Latrophilin3 by a synthetic binder},

author = {Szymon P Kordon and Przemysław Dutka and Justyna M Adamska and Sumit J Bandekar and Katherine Leon and Satchal K Erramilli and Brock Adams and Jingxian Li and Anthony A Kossiakoff and Demet Araç},

doi = {10.1038/s41467-023-36312-7},

issn = {2041-1723},

year  = {2023},

date = {2023-02-01},

journal = {Nat Commun},

volume = {14},

number = {1},

pages = {635},

abstract = {Adhesion G protein-coupled receptors (aGPCRs) are cell-surface proteins with large extracellular regions that bind to multiple ligands to regulate key biological functions including neurodevelopment and organogenesis. Modulating a single function of a specific aGPCR isoform while affecting no other function and no other receptor is not trivial. Here, we engineered an antibody, termed LK30, that binds to the extracellular region of the aGPCR ADGRL3, and specifically acts as an agonist for ADGRL3 but not for its isoform, ADGRL1. The LK30/ADGRL3 complex structure revealed that the LK30 binding site on ADGRL3 overlaps with the binding site for an ADGRL3 ligand - teneurin. In cellular-adhesion assays, LK30 specifically broke the trans-cellular interaction of ADGRL3 with teneurin, but not with another ADGRL3 ligand - FLRT3. Our work provides proof of concept for the modulation of isoform- and ligand-specific aGPCR functions using unique tools, and thus establishes a foundation for the development of fine-tuned aGPCR-targeted therapeutics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35952760,

title = {Development, structure, and mechanism of synthetic antibodies that target claudin and Clostridium perfringens enterotoxin complexes},

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

doi = {10.1016/j.jbc.2022.102357},

issn = {1083-351X},

year  = {2022},

date = {2022-09-01},

journal = {J Biol Chem},

volume = {298},

number = {9},

pages = {102357},

abstract = {Strains of Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflicts humans and domesticated animals, causing prevalent gastrointestinal illnesses. CpE's C-terminal domain (cCpE) binds cell surface receptors, followed by a restructuring of its N-terminal domain to form a membrane-penetrating β-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are known receptors for CpE and also control the architecture and function of cell-cell contacts (tight junctions) that create barriers to intercellular molecular transport. CpE binding and assembly disables claudin barrier function and induces cytotoxicity via β-pore formation, disrupting gut homeostasis; however, a structural basis of this process and strategies to inhibit the claudin-CpE interactions that trigger it are both lacking. Here, we used a synthetic antigen-binding fragment (sFab) library to discover two sFabs that bind claudin-4 and cCpE complexes. We established these sFabs' mode of molecular recognition and binding properties and determined structures of each sFab bound to claudin-4-cCpE complexes using cryo-EM. The structures reveal that the sFabs bind a shared epitope, but conform distinctly, which explains their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in binding changes, validating the structures, and uncovering the sFab's targeting mechanism. From these insights, we generated a model for CpE's claudin-bound β-pore that predicted sFabs would not prevent cytotoxicity, which we then verified in vivo. Taken together, this work demonstrates the development and mechanism of claudin/cCpE-binding sFabs that provide a framework and strategy for obstructing claudin/CpE assembly to treat CpE-linked gastrointestinal diseases.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}