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cNMP Signaling in Human and Microbial Pathogens.
Cyclic nucleotides are vital intracellular signaling molecules across domain of life. The most extensively studied cyclic nucleotides are cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which act as secondary messengers in numerous signal transduction pathways, including G-protein coupled receptor (GPCR) signaling, and play vital role in cellular functions, physiology. Owning to central role of cNMP in cell functions, microbial pathogens have evolved effectors toxin to disrupt host cNMP signaling system and hijacks' host cellular machineries for survival and infection. During my doctoral and postdoctoral research, I investigated membrane adenylyl cyclases (AC8 and Cya) and nucleotidyl cyclase toxins (ExoY) from both human and microbial pathogens.
Overview of cAMP signaling in human and microbial pathogens. Mammalian membrane adenylyl cyclases are key component of GPCR signaling. Pseudomonas aeruginosa secrete nucleotidyl cyclase toxin ExoY via type III secretion systems, subverting host cNMP signaling to facilitate its survival inside the host.
Role of flexible and Unstructured Regions in Regulation of Adenylyl Cyclases
Membrane-inte adenylyl cyclases catalyse synthesis of cAMP from ATP. Owning to their central role in cellular signaling, their function is finely tuned with other signaling pathways. We show that N terminus, C1b and C2b domain of membrane ACs are highly flexible structures or intrinsically unstructured, and form platform for interaction with diverse regulatory partners (such as Calmodulin, G-proteins) (Khanppnavar, Schuster et al., 2024). ExoY and other nucleotidyl cyclase toxins also encompass such unstructured domains to allosterically regulate its enzymatic activity (Khanppnavar et al., 2018).
Hybrid model of AC8 in complex with Calmodulin and G-protein subunits derived from integrative structural biology.
Membrane Adenylyl Cyclases as Receptors for Yet-to-be-Identified Ligands
Membrane Adenylyl Cyclases as Receptors for Yet-to-be-Identified Ligands
Although the critical role of mammalian membrane adenylyl cyclase (AC) in cellular signaling is well established, the function of their transmembrane domains remains unclear. Our cryo-EM structures of mammalian AC8 and mycobacterial membrane adenylyl cyclases Cya (an evolutionary ancestor of membrane AC) showed putative negatively charged ligand binding pockets in extracellular side of these transmembrane domains. Mutating key residues within these pockets altered the stability and enzymatic activity of Cya, supporting the hypothesis that the transmembrane domain may function as a receptor for unidentified molecules (Mehta, Khanppnavar et al., 2022; Khanppnavar, Schuster et al., 2024).
Depiction of the structural differences in the TM domain and extracellular surfaces features in Mycobacterium tuberculosis Rv1625c/Cya (PDB ID: 7YZI) and mammalian adenylyl cyclase AC8 (PDB ID: 8BUZ). The extracellular side of the TM domain features two negatively charged pockets (Ext1 and Ext2) in Rv1625c/Cya, one partially negative charged pocket (Ext1) in AC8.
ExoY as Tool for Studying Non-Canonical cUMP Signaling
Owning to central role of cNMP in cellular functions, opportunist pathogen Pseudomonas aeruginosa use nucleotidyl cyclase toxin ExoY to disrupt host cNMP signaling to facilitate its invasion of host. This effector toxin not only contribute to virulence of this ubiquitous pathogen but is also interesting for its ability to produce non-canonical cyclic nucleotide cUMP. Unlike, cAMP/cGMP, the function cUMP and cCMP remains elusive for decades, and ExoY is excellent model for studying functions these nucleotide messenger. Our structural and biochemical analysis provided basis for its substrate specificity (Khanppnavar & Datta, 2018).
Schematic representation of the crystal structure of the nucleotidyl cyclase toxin ExoY (PDB ID: 5XNW) with docked magnesium ion and UTP, highlighting conserved motifs involved in NTP binding and catalysis.
Molecular Basis for Functional Divergence of Duffy Antigen Receptor
The Duffy antigen receptor (DARC) is a seven-transmembrane (7TM) protein predominantly found on the surface of red blood cells, forming the basis of the Duffy blood group system in humans. DARC plays a key role as the primary binding site for the malarial parasite Plasmodium vivax and pore-forming toxins secreted by Staphylococcus aureus. Here, we unravel the molecular mechanisms by which this 7TM receptor, a member of the GPCR family, functions without coupling to G-proteins or arrestins, instead utilizing non-canonical signal transduction pathways (Saha, Khanppnavar, Maharana et al., 2024).
The schematic illustrates the importance of the cleft formed by the cytoplasmic ends of TM5 and TM6 in G protein, GRK, and β-arrestin coupling (left). DARC exhibits the most promiscuous interaction with chemokines, leading to scavenging functions. CRS2 in DARC is not directly involved in ligand binding. DARC possesses relatively shorter TM5/TM6, exhibiting limited conformational changes as compared with the canonical GPCRs, thereby precluding the interactions with G protein, GRK, and β-arrestins (right)
References:
1. Saha S.*, Khanppnavar B.*, Maharana J.*, Kim H., Carino C. M. C., Carole Daly C., Houston S., Kumari P., Yadav P. N., Plouffe B., Inoue A., Chung K. Y., Banerjee R., Korkhov V., Shukla A. K. Molecular mechanism of distinct chemokine engagement and functional divergence of the human Duffy antigen receptor. Cell (2024). DOI: 10.1016/j.cell.2024.07.005 *Co-first authors
2. Holfeld A., Schuster D., Sesterhenn F., Stalder P., Haenseler W., Barrio-Hernandez I., Ghosh D., Nagel L., Khanppnavar B., Beltrao P., Korkhov V., Riek R., Souza N. D., Picotti P. Systematic identification of structure-specific protein-protein interactions using limited proteolysis–mass spectrometry. Molecular Systems Biology (2024). https://doi.org/10.1038/s44320-024-00037-6
3. Khanppnavar B.*, Schuster D.*, Lavriha P., Uliana F., Özel M., Ved Mehta V., Leitner A., Picotti P., Korkhov V. M. Regulatory sites of CaM-sensitive adenylyl cyclase AC8 revealed by cryo-EM and structural proteomics. EMBO reports (2024). https://doi.org/10.1038/s44319-024-00076-y *Co-first authors
4. Schuster D., Khanppnavar B., Kantarci I., Mehta V., Korkhov V. M Structural insights into membrane adenylyl cyclases, initiators of cAMP signaling. Trends in Biochemical Sciences (2023). https://doi.org/10.1016/j.tibs.2023.12.002
5. Mehta V.*, Khanppnavar B.*, Schuster D., Kantarci I., Vercellino I., Kosturanova A., Iype T., Stefanic S., Picotti P., Korkhov V. M. Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases. eLife (2022) 11:e77032. https://doi.org/10.7554/eLife.77032 *Co-first authors
6. Qi C., Lavriha P., Mehta V., Khanppnavar B., Mohammed I., Li Y., Lazaratos M., Schaefer J. V., Dreier B., Plückthun A., Bondar A. N., Dessauer C. W., Korkhov V. M. Structural basis of adenylyl cyclase 9 activation. Nature Communications (2022). https://doi.org/10.1038/s41467-022-28685-y
7. Khanppnavar B.*, Mehta V*., Qi C*., Korkhov V. Structure and function of adenylyl cyclases, key enzymes in cellular signaling. Current Opinion in Structural Biology (2020). https://doi.org/10.1016/j.sbi.2020.03.003 *Co-first authors
8. Khanppnavar B., Datta S. Crystal structure and substrate specificity of ExoY, a unique T3SS mediated secreted nucleotidyl cyclase toxin from Pseudomonas aeruginosa. BBA-General Subjects (2018). https://doi.org/10.1016/j.bbagen.2018.05.021
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