The Kossiakoff Group’s research interests are to provide a molecular understanding of how molecular recognition governs virtually all aspects of biological function. To study these issues our group employs a combination of X-ray crystallography and cryo-EM, site-directed mutagenesis, phage display and biophysical analysis. The Kossiakoff group has also pioneered a new technology called “chaperone-assisted” crystallography, which has facilitated the structural analyses of protein systems that had been totally recalcitrant to other approaches. The group has also been at the forefront of developing synthetic antibodies. These synthetic antibodies are much more powerful than traditional monoclonal antibodies and have the potential to completely replace them for uses in live cell imaging and proteomics.
Latest Publications
Lodwick J E; Shen R; Erramilli S; Xie Y; Roganowicz K; Kossiakoff A A; Zhao M
Structural Insights into the Roles of PARP4 and NAD in the Human Vault Cage Journal Article
In: Nature Communications, iss. 16, no. 6724, 2025, ISSN: 2692-8205.
@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 = {10.1038/s41467-025-61981-x},
issn = {2692-8205},
year = {2025},
date = {2025-07-21},
urldate = {2024-06-01},
journal = {Nature Communications},
number = {6724},
issue = {16},
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}
}
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.
Arina A; Arauz E; Masoumi E; Warzecha K W; Sääf A; Widło Ł; Slezak T; Zieminska A; Dudek K; Schaefer Z P; Lecka M; Usatyuk S; Weichselbaum R R; Kossiakoff A A
A universal chimeric antigen receptor (CAR)-fragment antibody binder (FAB) split system for cancer immunotherapy Journal Article
In: Sci Adv, vol. 11, no. 27, pp. eadv4937, 2025, ISSN: 2375-2548.
@article{pmid40614208,
title = {A universal chimeric antigen receptor (CAR)-fragment antibody binder (FAB) split system for cancer immunotherapy},
author = {Ainhoa Arina and Edwin Arauz and Elham Masoumi and Karolina W Warzecha and Annika Sääf and Łukasz Widło and Tomasz Slezak and Aleksandra Zieminska and Karolina Dudek and Zachary P Schaefer and Maria Lecka and Svitlana Usatyuk and Ralph R Weichselbaum and Anthony A Kossiakoff},
doi = {10.1126/sciadv.adv4937},
issn = {2375-2548},
year = {2025},
date = {2025-07-01},
urldate = {2025-07-01},
journal = {Sci Adv},
volume = {11},
number = {27},
pages = {eadv4937},
abstract = {Chimeric antigen receptor (CAR) T cell therapy has shown extraordinary results in treating hematological cancer but faces challenges like antigen loss, toxicity, and complex manufacturing. Universal and modular CAR constructs offer improved flexibility, safety, and cost-effectiveness over conventional CAR constructs. We present a CAR-fragment antibody binder (Fab) platform on the basis of an engineered protein G variant (GA1) and Fab scaffolds. Expression of GA1CAR on human CD8 T cells leads to antigen recognition and T cell effector function that can be modulated according to the affinity of the CAR for the Fab and of the Fab for the target. GA1CAR T cells can recognize multiple Fab-antigen pairs on breast and ovarian cancer cell lines. Adoptively transferred GA1CAR T cells control tumors in breast cancer xenograft models, and their targeting can be quickly redirected using different Fabs. This versatile "plug-and-play" CAR T platform has potential for application in personalized therapy, preventing antigen loss variant escape, decreasing toxicity, and increasing access.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Chimeric antigen receptor (CAR) T cell therapy has shown extraordinary results in treating hematological cancer but faces challenges like antigen loss, toxicity, and complex manufacturing. Universal and modular CAR constructs offer improved flexibility, safety, and cost-effectiveness over conventional CAR constructs. We present a CAR-fragment antibody binder (Fab) platform on the basis of an engineered protein G variant (GA1) and Fab scaffolds. Expression of GA1CAR on human CD8 T cells leads to antigen recognition and T cell effector function that can be modulated according to the affinity of the CAR for the Fab and of the Fab for the target. GA1CAR T cells can recognize multiple Fab-antigen pairs on breast and ovarian cancer cell lines. Adoptively transferred GA1CAR T cells control tumors in breast cancer xenograft models, and their targeting can be quickly redirected using different Fabs. This versatile "plug-and-play" CAR T platform has potential for application in personalized therapy, preventing antigen loss variant escape, decreasing toxicity, and increasing access.
Ogbu C P; de Las Alas M; Mandriota A M; Liu X; Kapoor S; Choudhury J; Ruma Y N; Goodman M C; Sanders C R; Gonen T; Kossiakoff A A; Duffey M E; Vecchio A J
Biophysical basis of tight junction barrier modulation by a pan-claudin-binding molecule Journal Article
In: PNAS Nexus, vol. 4, no. 6, pp. pgaf189, 2025, ISSN: 2752-6542.
@article{pmid40575703,
title = {Biophysical basis of tight junction barrier modulation by a pan-claudin-binding molecule},
author = {Chinemerem P Ogbu and Mason de Las Alas and Alexandria M Mandriota and Xiangdong Liu and Srajan Kapoor and Jagrity Choudhury and Yasmeen N Ruma and Michael C Goodman and Charles R Sanders and Tamir Gonen and Anthony A Kossiakoff and Michael E Duffey and Alex J Vecchio},
doi = {10.1093/pnasnexus/pgaf189},
issn = {2752-6542},
year = {2025},
date = {2025-06-01},
urldate = {2025-06-01},
journal = {PNAS Nexus},
volume = {4},
number = {6},
pages = {pgaf189},
abstract = {Claudins are a 27-member family of membrane proteins that form and fortify specialized cell contacts in endothelium and epithelium called tight junctions. Tight junctions restrict paracellular transport through 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 physicochemical intractability and small targetable surfaces. Here, we determine that a synthetic antibody fragment (sFab) that we developed binds with nanomolar affinity directly to 10 claudin subtypes and other distantly related claudin family members but not to other tight junction-localized membrane proteins. It does so by targeting the extracellular surfaces of claudins, which we verify by applying this sFab to a model intestinal epithelium and observe that it opens paracellular barriers comparable to a known, but application limited, tight junction modulating protein. This pan-claudin-binding molecule holds potential for both basic and translational applications as it is a probe of claudin and tight junction structure in vitro and in vivo and a tool to modulate the permeability of tight junctions broadly across tissue barriers.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Claudins are a 27-member family of membrane proteins that form and fortify specialized cell contacts in endothelium and epithelium called tight junctions. Tight junctions restrict paracellular transport through 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 physicochemical intractability and small targetable surfaces. Here, we determine that a synthetic antibody fragment (sFab) that we developed binds with nanomolar affinity directly to 10 claudin subtypes and other distantly related claudin family members but not to other tight junction-localized membrane proteins. It does so by targeting the extracellular surfaces of claudins, which we verify by applying this sFab to a model intestinal epithelium and observe that it opens paracellular barriers comparable to a known, but application limited, tight junction modulating protein. This pan-claudin-binding molecule holds potential for both basic and translational applications as it is a probe of claudin and tight junction structure in vitro and in vivo and a tool to modulate the permeability of tight junctions broadly across tissue barriers.
O'Leary K M; Slezak T; Kossiakoff A A
Conformation-specific synthetic intrabodies modulate mTOR signaling with subcellular spatial resolution Journal Article
In: Proc Natl Acad Sci U S A, vol. 122, no. 24, pp. e2424679122, 2025, ISSN: 1091-6490.
@article{pmid40489625,
title = {Conformation-specific synthetic intrabodies modulate mTOR signaling with subcellular spatial resolution},
author = {Kelly M O'Leary and Tomasz Slezak and Anthony A Kossiakoff},
doi = {10.1073/pnas.2424679122},
issn = {1091-6490},
year = {2025},
date = {2025-06-01},
urldate = {2025-06-01},
journal = {Proc Natl Acad Sci U S A},
volume = {122},
number = {24},
pages = {e2424679122},
abstract = {Subcellular compartmentalization is integral to the spatial regulation of mechanistic target of rapamycin (mTOR) signaling. However, the biological outputs associated with location-specific mTOR signaling events are poorly understood and challenging to decouple. Here, we engineered synthetic intracellular antibodies (intrabodies) that are capable of modulating mTOR signaling with genetically programmable spatial resolution. Epitope-directed phage display was exploited to generate high affinity synthetic antibody fragments (Fabs) against the FKBP12-Rapamycin binding site of mTOR (mTOR). We determined high-resolution crystal structures of two unique Fabs that discriminate distinct conformational states of mTOR through recognition of its substrate recruitment interface. By leveraging these conformation-specific binders as intracellular probes, we uncovered the structural basis for an allosteric mechanism governing mTOR complex 1 (mTORC1) stability mediated by subtle structural adjustments within mTOR. Furthermore, our results demonstrated that synthetic binders emulate natural substrates by employing divergent yet complementary hydrophobic residues at defined positions, underscoring the broad molecular recognition capability of mTOR. Intracellular signaling studies showed differential time-dependent inhibition of S6 kinase 1 and Akt phosphorylation by genetically encoded intrabodies, thus supporting a mechanism of inhibition analogous to the natural product rapamycin. Finally, we implemented a feasible approach to selectively modulate mTOR signaling in the nucleus through spatially programmed intrabody expression. These findings establish intrabodies as versatile tools for dissecting the conformational regulation of mTORC1 and should be useful to explore how location-specific mTOR signaling influences disease progression.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Subcellular compartmentalization is integral to the spatial regulation of mechanistic target of rapamycin (mTOR) signaling. However, the biological outputs associated with location-specific mTOR signaling events are poorly understood and challenging to decouple. Here, we engineered synthetic intracellular antibodies (intrabodies) that are capable of modulating mTOR signaling with genetically programmable spatial resolution. Epitope-directed phage display was exploited to generate high affinity synthetic antibody fragments (Fabs) against the FKBP12-Rapamycin binding site of mTOR (mTOR). We determined high-resolution crystal structures of two unique Fabs that discriminate distinct conformational states of mTOR through recognition of its substrate recruitment interface. By leveraging these conformation-specific binders as intracellular probes, we uncovered the structural basis for an allosteric mechanism governing mTOR complex 1 (mTORC1) stability mediated by subtle structural adjustments within mTOR. Furthermore, our results demonstrated that synthetic binders emulate natural substrates by employing divergent yet complementary hydrophobic residues at defined positions, underscoring the broad molecular recognition capability of mTOR. Intracellular signaling studies showed differential time-dependent inhibition of S6 kinase 1 and Akt phosphorylation by genetically encoded intrabodies, thus supporting a mechanism of inhibition analogous to the natural product rapamycin. Finally, we implemented a feasible approach to selectively modulate mTOR signaling in the nucleus through spatially programmed intrabody expression. These findings establish intrabodies as versatile tools for dissecting the conformational regulation of mTORC1 and should be useful to explore how location-specific mTOR signaling influences disease progression.
Chen Q; Schafer C T; Mukherjee S; Wang K; Gustavsson M; Fuller J R; Tepper K; Lamme T D; Aydin Y; Agrawal P; Terashi G; Yao X; Kihara D; Kossiakoff A A; Handel T M; Tesmer J J G
Effect of phosphorylation barcodes on arrestin binding to a chemokine receptor Journal Article
In: Nature, 2025, ISSN: 1476-4687.
@article{pmid40399676,
title = {Effect of phosphorylation barcodes on arrestin binding to a chemokine receptor},
author = {Qiuyan Chen and Christopher T Schafer and Somnath Mukherjee and Kai Wang and Martin Gustavsson and James R Fuller and Katelyn Tepper and Thomas D Lamme and Yasmin Aydin and Parth Agrawal and Genki Terashi and Xin-Qiu Yao and Daisuke Kihara and Anthony A Kossiakoff and Tracy M Handel and John J G Tesmer},
doi = {10.1038/s41586-025-09024-9},
issn = {1476-4687},
year = {2025},
date = {2025-05-01},
urldate = {2025-05-01},
journal = {Nature},
abstract = {Unique phosphorylation 'barcodes' installed in different regions of an active seven-transmembrane receptor by different G-protein-coupled receptor (GPCR) kinases (GRKs) have been proposed to promote distinct cellular outcomes, but it is unclear whether or how arrestins differentially engage these barcodes. Here, to address this, we developed an antigen-binding fragment (Fab7) that recognizes both active arrestin2 (β-arrestin1) and arrestin3 (β-arrestin2) without interacting with bound receptor polypeptides. We used Fab7 to determine the structures of both arrestins in complex with atypical chemokine receptor 3 (ACKR3) phosphorylated in different regions of its C-terminal tail by either GRK2 or GRK5 (ref. ). The GRK2-phosphorylated ACKR3 resulted in more heterogeneous 'tail-mode' assemblies, whereas phosphorylation by GRK5 resulted in more rigid 'ACKR3-adjacent' assemblies. Unexpectedly, the finger loops of both arrestins engaged the micelle surface rather than the receptor intracellular pocket, with arrestin3 being more dynamic, partly because of its lack of a membrane-anchoring motif. Thus, both the region of the barcode and the arrestin isoform involved can alter the structure and dynamics of GPCR-arrestin complexes, providing a possible mechanistic basis for unique downstream cellular effects, such as the efficiency of chemokine scavenging and the robustness of arrestin binding in ACKR3.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Unique phosphorylation 'barcodes' installed in different regions of an active seven-transmembrane receptor by different G-protein-coupled receptor (GPCR) kinases (GRKs) have been proposed to promote distinct cellular outcomes, but it is unclear whether or how arrestins differentially engage these barcodes. Here, to address this, we developed an antigen-binding fragment (Fab7) that recognizes both active arrestin2 (β-arrestin1) and arrestin3 (β-arrestin2) without interacting with bound receptor polypeptides. We used Fab7 to determine the structures of both arrestins in complex with atypical chemokine receptor 3 (ACKR3) phosphorylated in different regions of its C-terminal tail by either GRK2 or GRK5 (ref. ). The GRK2-phosphorylated ACKR3 resulted in more heterogeneous 'tail-mode' assemblies, whereas phosphorylation by GRK5 resulted in more rigid 'ACKR3-adjacent' assemblies. Unexpectedly, the finger loops of both arrestins engaged the micelle surface rather than the receptor intracellular pocket, with arrestin3 being more dynamic, partly because of its lack of a membrane-anchoring motif. Thus, both the region of the barcode and the arrestin isoform involved can alter the structure and dynamics of GPCR-arrestin complexes, providing a possible mechanistic basis for unique downstream cellular effects, such as the efficiency of chemokine scavenging and the robustness of arrestin binding in ACKR3.
Chen L; Dickerhoff J; Zheng K; Erramilli S; Feng H; Wu G; Onel B; Chen Y; Wang K; Carver M; Lin C; Sakai S; Wan J; Vinson C; Hurley L; Kossiakoff A A; Deng N; Bai Y; Noinaj N; Yang D
Structural basis for nucleolin recognition of MYC promoter G-quadruplex Journal Article
In: Science, vol. 388, no. 6744, 2025, ISSN: 1095-9203.
@article{Chen2025,
title = {Structural basis for nucleolin recognition of MYC promoter G-quadruplex},
author = {Luying Chen and Jonathan Dickerhoff and Ke-wei Zheng and Satchal Erramilli and Hanqiao Feng and Guanhui Wu and Buket Onel and Yuwei Chen and Kai-Bo Wang and Megan Carver and Clement Lin and Saburo Sakai and Jun Wan and Charles Vinson and Laurence Hurley and Anthony A. Kossiakoff and Nanjie Deng and Yawen Bai and Nicholas Noinaj and Danzhou Yang},
doi = {10.1126/science.adr1752},
issn = {1095-9203},
year = {2025},
date = {2025-04-18},
urldate = {2025-04-18},
journal = {Science},
volume = {388},
number = {6744},
publisher = {American Association for the Advancement of Science (AAAS)},
abstract = {The MYC oncogene promoter G-quadruplex (MycG4) regulates transcription and is a prevalent G4 locus in immortal cells. Nucleolin, a major MycG4-binding protein, exhibits greater affinity for MycG4 than for nucleolin recognition element (NRE) RNA. Nucleolin’s four RNA binding domains (RBDs) are essential for high-affinity MycG4 binding. We present the 2.6-angstrom crystal structure of the nucleolin-MycG4 complex, revealing a folded parallel three-tetrad G-quadruplex with two coordinating potassium ions (K+), interacting with RBD1, RBD2, and Linker12 through its 6–nucleotide (nt) central loop and 5′ flanking region. RBD3 and RBD4 bind MycG4’s 1-nt loops as demonstrated by nuclear magnetic resonance (NMR). Cleavage under targets and tagmentation sequencing confirmed nucleolin’s binding to MycG4 in cells. Our results revealed a G4 conformation-based recognition by a regulating protein through multivalent interactions, suggesting that G4s are nucleolin’s primary cellular substrates, indicating G4 epigenetic transcriptional regulation and helping G4-targeted drug discovery.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The MYC oncogene promoter G-quadruplex (MycG4) regulates transcription and is a prevalent G4 locus in immortal cells. Nucleolin, a major MycG4-binding protein, exhibits greater affinity for MycG4 than for nucleolin recognition element (NRE) RNA. Nucleolin’s four RNA binding domains (RBDs) are essential for high-affinity MycG4 binding. We present the 2.6-angstrom crystal structure of the nucleolin-MycG4 complex, revealing a folded parallel three-tetrad G-quadruplex with two coordinating potassium ions (K+), interacting with RBD1, RBD2, and Linker12 through its 6–nucleotide (nt) central loop and 5′ flanking region. RBD3 and RBD4 bind MycG4’s 1-nt loops as demonstrated by nuclear magnetic resonance (NMR). Cleavage under targets and tagmentation sequencing confirmed nucleolin’s binding to MycG4 in cells. Our results revealed a G4 conformation-based recognition by a regulating protein through multivalent interactions, suggesting that G4s are nucleolin’s primary cellular substrates, indicating G4 epigenetic transcriptional regulation and helping G4-targeted drug discovery.