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
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.
Ramesh A; Roy S; Slezak T; Fuller J; Graves H; Mamedov M R; Marson A; Kossiakoff A A; Adams E J
Mapping the extracellular molecular architecture of the pAg-signaling complex with α-Butyrophilin antibodies Journal Article
In: Sci Rep, vol. 15, no. 1, 2025, ISSN: 2045-2322.
@article{Ramesh2025,
title = {Mapping the extracellular molecular architecture of the pAg-signaling complex with α-Butyrophilin antibodies},
author = {Amrita Ramesh and Sobhan Roy and Tomasz Slezak and James Fuller and Hortencia Graves and Murad R. Mamedov and Alexander Marson and Anthony A. Kossiakoff and Erin J. Adams},
doi = {10.1038/s41598-025-94347-w},
issn = {2045-2322},
year = {2025},
date = {2025-04-09},
urldate = {2025-12-00},
journal = {Sci Rep},
volume = {15},
number = {1},
publisher = {Springer Science and Business Media LLC},
abstract = {Target cells trigger Vγ9Vδ2 T cell activation by signaling the intracellular accumulation of phospho-antigen metabolites (pAgs) through Butyrophilin (BTN)-3A1 and BTN2A1 to the Vγ9Vδ2 T cell receptor (TCR). An incomplete understanding of the molecular dynamics in this signaling complex hampers Vγ9Vδ2 T cell immunotherapeutic efficacy. A panel of engineered α-BTN3A1 and α-BTN2A1 antibody (mAb) reagents was used to probe the roles of BTN3A1 and BTN2A1 in pAg signaling. Modified α-BTN3A1 mAbs with increased inter-Fab distances establish that tight clustering of BTN3A1 is not necessary to stimulate Vγ9Vδ2 T cell activation, and that antagonism may occur through occlusion of a critical binding interaction between BTN3A1 and a yet unknown co-receptor. Finally, a panel of additional α-BTN2A1 antagonists utilize different biophysical mechanisms to compete with Vγ9Vδ2 TCRs for BTN2A1 binding. The complex structures of BTN2A1 ectodomain and Fabs from three antagonist mAbs provide molecular insights into BTN2A1 epitopes critical for pAg-signaling.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Target cells trigger Vγ9Vδ2 T cell activation by signaling the intracellular accumulation of phospho-antigen metabolites (pAgs) through Butyrophilin (BTN)-3A1 and BTN2A1 to the Vγ9Vδ2 T cell receptor (TCR). An incomplete understanding of the molecular dynamics in this signaling complex hampers Vγ9Vδ2 T cell immunotherapeutic efficacy. A panel of engineered α-BTN3A1 and α-BTN2A1 antibody (mAb) reagents was used to probe the roles of BTN3A1 and BTN2A1 in pAg signaling. Modified α-BTN3A1 mAbs with increased inter-Fab distances establish that tight clustering of BTN3A1 is not necessary to stimulate Vγ9Vδ2 T cell activation, and that antagonism may occur through occlusion of a critical binding interaction between BTN3A1 and a yet unknown co-receptor. Finally, a panel of additional α-BTN2A1 antagonists utilize different biophysical mechanisms to compete with Vγ9Vδ2 TCRs for BTN2A1 binding. The complex structures of BTN2A1 ectodomain and Fabs from three antagonist mAbs provide molecular insights into BTN2A1 epitopes critical for pAg-signaling.
Liu Y; Brown C M; Erramilli S; Su Y; Tseng P; Wang Y; Duong N H; Tokarz P; Kloss B; Han C; Chen H; Rodrigues J; Archer M; Lowary T L; Kossiakoff A A; Stansfeld P J; Nygaard R; Mancia F
Structural insights into terminal arabinosylation biosynthesis of the mycobacterial cell wall arabinan Journal Article
In: Nature Communications, 2025, ISSN: 2692-8205.
@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 = {10.1038/s41467-025-58196-5},
issn = {2692-8205},
year = {2025},
date = {2025-03-14},
urldate = {2024-09-01},
journal = {Nature Communications},
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}
}
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.
Srivastava S; Sekar G; Ojoawo A; Aggarwal A; Ferreira E; Uchikawa E; Yang M; Grace C R; Dey R; Lin Y; Guibao C D; Jayaraman S; Mukherjee S; Kossiakoff A A; Dong B; Myasnikov A; Moldoveanu T
Structural basis of BAK sequestration by MCL-1 in apoptosis Journal Article
In: Mol Cell, 2025, ISSN: 1097-4164.
@article{pmid40187349,
title = {Structural basis of BAK sequestration by MCL-1 in apoptosis},
author = {Shagun Srivastava and Giridhar Sekar and Adedolapo Ojoawo and Anup Aggarwal and Elisabeth Ferreira and Emiko Uchikawa and Meek Yang and Christy R Grace and Raja Dey and Yi-Lun Lin and Cristina D Guibao and Seetharaman Jayaraman and Somnath Mukherjee and Anthony A Kossiakoff and Bin Dong and Alexander Myasnikov and Tudor Moldoveanu},
doi = {10.1016/j.molcel.2025.03.013},
issn = {1097-4164},
year = {2025},
date = {2025-03-01},
urldate = {2025-03-01},
journal = {Mol Cell},
abstract = {Apoptosis controls cell fate, ensuring tissue homeostasis and promoting disease when dysregulated. The rate-limiting step in apoptosis is mitochondrial poration by the effector B cell lymphoma 2 (BCL-2) family proteins BAK and BAX, which are activated by initiator BCL-2 homology 3 (BH3)-only proteins (e.g., BIM) and inhibited by guardian BCL-2 family proteins (e.g., MCL-1). We integrated structural, biochemical, and pharmacological approaches to characterize the human prosurvival MCL-1:BAK complex assembled from their BCL-2 globular core domains. We reveal a canonical interaction with BAK BH3 bound to the hydrophobic groove of MCL-1 and disordered and highly dynamic BAK regions outside the complex interface. We predict similar conformations of activated effectors in complex with other guardians or effectors. The MCL-1:BAK complex is a major cancer drug target. We show that MCL-1 inhibitors are inefficient in neutralizing the MCL-1:BAK complex, requiring high doses to initiate apoptosis. Our study underscores the need to design superior clinical candidate MCL-1 inhibitors.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Apoptosis controls cell fate, ensuring tissue homeostasis and promoting disease when dysregulated. The rate-limiting step in apoptosis is mitochondrial poration by the effector B cell lymphoma 2 (BCL-2) family proteins BAK and BAX, which are activated by initiator BCL-2 homology 3 (BH3)-only proteins (e.g., BIM) and inhibited by guardian BCL-2 family proteins (e.g., MCL-1). We integrated structural, biochemical, and pharmacological approaches to characterize the human prosurvival MCL-1:BAK complex assembled from their BCL-2 globular core domains. We reveal a canonical interaction with BAK BH3 bound to the hydrophobic groove of MCL-1 and disordered and highly dynamic BAK regions outside the complex interface. We predict similar conformations of activated effectors in complex with other guardians or effectors. The MCL-1:BAK complex is a major cancer drug target. We show that MCL-1 inhibitors are inefficient in neutralizing the MCL-1:BAK complex, requiring high doses to initiate apoptosis. Our study underscores the need to design superior clinical candidate MCL-1 inhibitors.