Structural Dynamics and Isoform-Specific Modulation of Human Adenylyl Cyclases
EMBARGOED UNTIL 2028-12-02
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2025
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Doctoral Thesis
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EMBARGOED UNTIL 2028-12-02
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Abstract
Transmembrane adenylyl cyclases (tmACs) are integral membrane proteins that catalyze the conversion of ATP into cAMP, a key second messenger in eukaryotic signaling. The nine mammalian isoforms are grouped into four classes based on their distinct regulatory responses to G protein signaling. Adenylyl Cyclase 9 (AC9) is the only member of Group 4, and, to date, Gαs is the only direct activator identified for this isoform. AC9 is highly expressed in the lung, heart, and skeletal muscle, and its dysregulation has been associated with pathological conditions such as asthma, bradycardia, and colon cancer. Despite recent structural insights, the molecular mechanisms underlying AC9’s catalytic activity and isoform-specific regulation remain poorly understood.
The primary objective of my doctoral thesis was to define the structural and mechanistic principles of AC9 regulation through biochemical and structural methods, with the long-term goal of enabling selective targeting of AC9 and other tmAC isoforms.
In the first part of this study, I investigated the potential regulatory interactions of AC9 using a combination of in vitro biochemical assays and structural analyses. However, no stable complexes were detected, indicating that these candidate regulators may not directly interact with AC9 or that additional cellular components are required for stable complex formation. I next focused on AC2, a canonical transmembrane adenylyl cyclase (tmAC) isoform synergistically activated by Gαs and Gβγ subunits, to further investigate G protein–mediated regulation. Through systematic optimization of AC2 expression and cryo-EM sample preparation, I established a robust purification workflow and successfully reconstituted the AC2–Gαsβγ complex. Although the resulting cryo-EM reconstruction was obtained at relatively low resolution, it provides an important foundation for future high-resolution structural studies.
In the second part of the study, I determined the cryo-EM structure of human AC9, which revealed multiple conformational states. One state was occluded, while another was ATPαs-bound, providing key mechanistic insights into AC9's conformational dynamics and its role in regulating catalytic activity. Guided by these observations, I used a structure-based chimeric design strategy, replacing elements of the AC9 catalytic site with those from AC5, an isoform more responsive to some nucleotide analogs. Analysis of the resulting AC95 chimera showed that forskolin binding induced a distinct conformation of the ATPαs site compared to native AC9. This approach enabled the dissection of allosteric and catalytic contributions to enzyme regulation, demonstrating how structural flexibility mediates enzymatic output and ligand responsiveness in ACs.
In conclusion, these findings advance our understanding of conformational dynamics, allosteric control, and isoform-specific regulation in tmACs, providing a foundation for the development of selective modulators targeting dysregulated cAMP signaling in disease.
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ETH Zurich
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09529 - Korkhov, Volodymyr / Korkhov, Volodymyr