Our interests are to determine the structure/function relationships driving biological process. We are particularly interested in the molecular recognition events that lead to ligand- induced receptor activation and regulation. We also have programs in structure determination of membrane proteins and multi-protein complexes.
Latest Publications
Slezak T; O'Leary K M; Li J; Rohaim A; Davydova E K; Kossiakoff A A
Engineered protein G variants for multifunctional antibody-based assemblies Journal Article
In: Protein Sci, vol. 34, no. 2, pp. e70019, 2025, ISSN: 1469-896X.
@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}
}
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.
Górniak I; Stephens Z; Erramilli S K; Gawda T; Kossiakoff A A; Zimmer J
Structural insights into translocation and tailored synthesis of hyaluronan Journal Article
In: Nat Struct Mol Biol, vol. 32, no. 1, pp. 161–171, 2025, ISSN: 1545-9985.
@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}
}
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.
Kordon S P; Cechova K; Bandekar S J; Leon K; Dutka P; Siffer G; Kossiakoff A A; Vafabakhsh R; Araç D
Conformational coupling between extracellular and transmembrane domains modulates holo-adhesion GPCR function Journal Article
In: Nat Commun, vol. 15, no. 1, pp. 10545, 2024, ISSN: 2041-1723.
@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}
}
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.
Rathnayake S S; Erramilli S K; Kossiakoff A A; Vecchio A J
Cryo-EM structures of Clostridium perfringens enterotoxin bound to its human receptor, claudin-4 Journal Article
In: Structure, vol. 32, no. 11, pp. 1936–1951.e5, 2024, ISSN: 1878-4186.
@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}
}
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.
Ogbu C P; Mandriota A M; Liu X; de Las Alas M; Kapoor S; Choudhury J; Kossiakoff A A; Duffey M E; Vecchio A J
Biophysical Basis of Paracellular Barrier Modulation by a Pan-Claudin-Binding Molecule Journal Article
In: bioRxiv, 2024, ISSN: 2692-8205.
@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}
}
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.
Ross P; Hilton H G; Lodwick J; Slezak T; Guethlein L A; McMurtrey C P; Han A S; Nielsen M; Yong D; Dulberger C L; Nolan K T; Roy S; Castro C D; Hildebrand W H; Zhao M; Kossiakoff A; Parham P; Adams E J
Molecular characterization of the archaic HLA-B*73:01 allele reveals presentation of a unique peptidome and skewed engagement by KIR2DL2 Journal Article
In: bioRxiv, 2024, ISSN: 2692-8205.
@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}
}
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.