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
Zinkle A P; Batista M B; Herrera C M; Erramilli S K; Kloss B; Ashraf K U; Nosol K; Zhang G; Cater R J; Marty M T; Kossiakoff A A; Trent M S; Nygaard R; Stansfeld P J; Mancia F
Mechanistic basis of antimicrobial resistance mediated by the phosphoethanolamine transferase MCR-1 Journal Article
In: Nat Commun, vol. 16, no. 1, pp. 10516, 2025, ISSN: 2041-1723.
@article{pmid41298376,
title = {Mechanistic basis of antimicrobial resistance mediated by the phosphoethanolamine transferase MCR-1},
author = {Allen P Zinkle and Mariana Bunoro Batista and Carmen M Herrera and Satchal K Erramilli and Brian Kloss and Khuram U Ashraf and Kamil Nosol and Guozhi Zhang and Rosemary J Cater and Michael T Marty and Anthony A Kossiakoff and M Stephen Trent and Rie Nygaard and Phillip J Stansfeld and Filippo Mancia},
doi = {10.1038/s41467-025-65515-3},
issn = {2041-1723},
year = {2025},
date = {2025-11-01},
urldate = {2025-11-01},
journal = {Nat Commun},
volume = {16},
number = {1},
pages = {10516},
abstract = {Polymyxins are used to treat infections caused by multidrug-resistant Gram-negative bacteria. They are cationic peptides that target the negatively charged lipid A component of lipopolysaccharides, disrupting the outer membrane and lysing the cell. Polymyxin resistance is conferred by inner-membrane enzymes, such as phosphoethanolamine transferases, which add positively charged phosphoethanolamine to lipid A. Here, we present the structure of MCR-1, a plasmid-encoded phosphoethanolamine transferase, in its liganded form. The phosphatidylethanolamine donor substrate is bound near the active site in the periplasmic domain, and lipid A is bound over 20 Å away, within the transmembrane region. Integrating structural, biochemical, and drug-resistance data with computational analyses, we propose a two-state model in which the periplasmic domain rotates to bring the active site to lipid A, near the preferential phosphate modification site for MCR-1. This enzymatic mechanism may be generally applicable to other phosphoform transferases with large, globular soluble domains.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Polymyxins are used to treat infections caused by multidrug-resistant Gram-negative bacteria. They are cationic peptides that target the negatively charged lipid A component of lipopolysaccharides, disrupting the outer membrane and lysing the cell. Polymyxin resistance is conferred by inner-membrane enzymes, such as phosphoethanolamine transferases, which add positively charged phosphoethanolamine to lipid A. Here, we present the structure of MCR-1, a plasmid-encoded phosphoethanolamine transferase, in its liganded form. The phosphatidylethanolamine donor substrate is bound near the active site in the periplasmic domain, and lipid A is bound over 20 Å away, within the transmembrane region. Integrating structural, biochemical, and drug-resistance data with computational analyses, we propose a two-state model in which the periplasmic domain rotates to bring the active site to lipid A, near the preferential phosphate modification site for MCR-1. This enzymatic mechanism may be generally applicable to other phosphoform transferases with large, globular soluble domains.
Romane K; Peteani G; Mukherjee S; Kowal J; Rossi L; Hou J; Kossiakoff A A; Lemmin T; Locher K P
Structural basis of drug recognition by human MATE1 transporter Journal Article
In: Nat Commun, vol. 16, no. 1, pp. 9444, 2025, ISSN: 2041-1723.
@article{pmid41145429,
title = {Structural basis of drug recognition by human MATE1 transporter},
author = {Ksenija Romane and Giulia Peteani and Somnath Mukherjee and Julia Kowal and Lorenzo Rossi and Jingkai Hou and Anthony A Kossiakoff and Thomas Lemmin and Kaspar P Locher},
doi = {10.1038/s41467-025-64490-z},
issn = {2041-1723},
year = {2025},
date = {2025-10-28},
urldate = {2025-10-01},
journal = {Nat Commun},
volume = {16},
number = {1},
pages = {9444},
abstract = {Human MATE1 (multidrug and toxin extrusion protein 1) is highly expressed in the kidney and liver, where it mediates the final step in the excretion of a broad range of cationic drugs, including the antidiabetic drug metformin, into the urine and bile. This transport process is essential for drug clearance and also affects therapeutic efficacy. To understand the molecular basis of drug recognition by hMATE1, we determined cryo-electron microscopy structures of the transporter in complex with the substrates 1-methyl-4-phenylpyridinium (MPP) and metformin and with the inhibitor cimetidine. The structures reveal a shared binding site located in a negatively charged pocket in the C-lobe of the protein. We functionally validated key interactions using radioactivity-based cellular uptake assays using hMATE1 mutants. Molecular dynamics simulations provide insights into the different binding modes and dynamic behaviour of the ligands within the pocket. Collectively, these findings define the structural basis of hMATE1 substrate specificity and shed light on its role in drug transport and drug-drug interactions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Human MATE1 (multidrug and toxin extrusion protein 1) is highly expressed in the kidney and liver, where it mediates the final step in the excretion of a broad range of cationic drugs, including the antidiabetic drug metformin, into the urine and bile. This transport process is essential for drug clearance and also affects therapeutic efficacy. To understand the molecular basis of drug recognition by hMATE1, we determined cryo-electron microscopy structures of the transporter in complex with the substrates 1-methyl-4-phenylpyridinium (MPP) and metformin and with the inhibitor cimetidine. The structures reveal a shared binding site located in a negatively charged pocket in the C-lobe of the protein. We functionally validated key interactions using radioactivity-based cellular uptake assays using hMATE1 mutants. Molecular dynamics simulations provide insights into the different binding modes and dynamic behaviour of the ligands within the pocket. Collectively, these findings define the structural basis of hMATE1 substrate specificity and shed light on its role in drug transport and drug-drug interactions.
Mohona S; Shakya A K; Singh S; Kearns F L; Jemison K; Erramilli S; Dey D; Qing E; Jennings B C; Doray B; Kossiakoff A A; Amaro R E; Klose T; Gallagher T; Hasan S S
An unconventional HxD motif orchestrates coatomer-dependent coronavirus morphogenesis Journal Article
In: bioRxiv, 2025, ISSN: 2692-8205.
@article{pmid41279991,
title = {An unconventional HxD motif orchestrates coatomer-dependent coronavirus morphogenesis},
author = {Surovi Mohona and Anil K Shakya and Suruchi Singh and Fiona L Kearns and Kezia Jemison and Satchal Erramilli and Debajit Dey and Enya Qing and Benjamin C Jennings and Balraj Doray and Anthony A Kossiakoff and Rommie E Amaro and Thomas Klose and Tom Gallagher and S Saif Hasan},
doi = {10.1101/2025.10.16.682669},
issn = {2692-8205},
year = {2025},
date = {2025-10-17},
urldate = {2025-10-01},
journal = {bioRxiv},
abstract = {Assembly of infectious coronaviruses requires spike (S) protein trafficking by host coatomer, typically via a dibasic signal in the S cytoplasmic tail. However, the human embecoviruses HKU1 and OC43, as well as the model virus MHV, lack this motif. Here we identify a conserved His-x-Asp (HxD) sequence that functions as an unconventional coatomer-binding signal. Structural and biochemical analyses show that the MHV HxD motif engages coatomer subunits through distinct conformations, while cellular imaging demonstrates its role in directing S to assembly sites with the viral M-protein. Disruption of HxD-coatomer interactions impairs S incorporation and provokes compensatory viral adaptations, including emergence of a canonical dibasic motif or mutations in M-protein. Electron microscopy further reveals profound alterations in virion surface architecture. These findings uncover HxD as a previously unrecognized coatomer-targeting motif, highlighting an unexpected flexibility in coronavirus assembly pathways and broadening understanding of the cellular machinery that shapes coronavirus morphogenesis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Assembly of infectious coronaviruses requires spike (S) protein trafficking by host coatomer, typically via a dibasic signal in the S cytoplasmic tail. However, the human embecoviruses HKU1 and OC43, as well as the model virus MHV, lack this motif. Here we identify a conserved His-x-Asp (HxD) sequence that functions as an unconventional coatomer-binding signal. Structural and biochemical analyses show that the MHV HxD motif engages coatomer subunits through distinct conformations, while cellular imaging demonstrates its role in directing S to assembly sites with the viral M-protein. Disruption of HxD-coatomer interactions impairs S incorporation and provokes compensatory viral adaptations, including emergence of a canonical dibasic motif or mutations in M-protein. Electron microscopy further reveals profound alterations in virion surface architecture. These findings uncover HxD as a previously unrecognized coatomer-targeting motif, highlighting an unexpected flexibility in coronavirus assembly pathways and broadening understanding of the cellular machinery that shapes coronavirus morphogenesis.
Cottom C O; Heinz E; Erramilli S; Kossiakoff A; Slade D J; Noinaj N
Characterization of the OMP biogenesis machinery in Fusobacterium nucleatum Journal Article
In: Structure, vol. 33, iss. 33, no. 11, pp. 1878–1892.e5, 2025, ISSN: 1878-4186.
@article{pmid40897170,
title = {Characterization of the OMP biogenesis machinery in Fusobacterium nucleatum},
author = {Claire Overly Cottom and Eva Heinz and Satchal Erramilli and Anthony Kossiakoff and Daniel J Slade and Nicholas Noinaj},
doi = {10.1016/j.str.2025.08.008},
issn = {1878-4186},
year = {2025},
date = {2025-09-01},
urldate = {2025-09-01},
journal = {Structure},
volume = {33},
number = {11},
issue = {33},
pages = {1878--1892.e5},
abstract = {F. nucleatum is a Gram-negative bacteria that causes oral infections and is linked to colorectal cancer. Pathogenicity relies on a type of β-barrel outer membrane protein (OMP) called an autotransporter. The biogenesis of OMPs is typically mediated by the barrel assembly machinery (BAM) complex. In this study, we investigate the evolution, composition, and structure of the OMP biogenesis machinery in F. nucleatum. Our bioinformatics and proteomics analyses indicate that OMP biogenesis in F. nucleatum is mediated solely by the core component BamA. The structure of FnBamA highlights distinct features, including four POTRA domains and a C-terminal 16-stranded β-barrel domain observed as an inverted dimer. FnBamA represents the original composition of the assembly machinery, and a duplication event that resulted in BamA and TamA occurred after the split of other lineages, including the Proteobacteria, from the Fusobacteria. FnBamA, therefore, likely serves a singular role in the biogenesis of all OMPs.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
F. nucleatum is a Gram-negative bacteria that causes oral infections and is linked to colorectal cancer. Pathogenicity relies on a type of β-barrel outer membrane protein (OMP) called an autotransporter. The biogenesis of OMPs is typically mediated by the barrel assembly machinery (BAM) complex. In this study, we investigate the evolution, composition, and structure of the OMP biogenesis machinery in F. nucleatum. Our bioinformatics and proteomics analyses indicate that OMP biogenesis in F. nucleatum is mediated solely by the core component BamA. The structure of FnBamA highlights distinct features, including four POTRA domains and a C-terminal 16-stranded β-barrel domain observed as an inverted dimer. FnBamA represents the original composition of the assembly machinery, and a duplication event that resulted in BamA and TamA occurred after the split of other lineages, including the Proteobacteria, from the Fusobacteria. FnBamA, therefore, likely serves a singular role in the biogenesis of all OMPs.
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.