Biophysical analysis of the membrane-proximal Venus Flytrap domain of ESAG4 receptor-like adenylate cyclase from Trypanosoma brucei.

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  • Additional Information
    • Source:
      Publisher: Elsevier/North-Holland Biomedical Press Country of Publication: Netherlands NLM ID: 8006324 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1872-9428 (Electronic) Linking ISSN: 01666851 NLM ISO Abbreviation: Mol Biochem Parasitol Subsets: MEDLINE
    • Publication Information:
      Original Publication: Amsterdam, Elsevier/North-Holland Biomedical Press.
    • Subject Terms:
      Trypanosoma brucei brucei*/enzymology ; Trypanosoma brucei brucei*/genetics ; Adenylyl Cyclases*/genetics ; Adenylyl Cyclases*/chemistry ; Adenylyl Cyclases*/metabolism; Protein Domains ; Protozoan Proteins/genetics ; Protozoan Proteins/chemistry ; Protozoan Proteins/metabolism ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; Recombinant Proteins/chemistry ; Models, Molecular
    • Abstract:
      The protozoan parasite Trypanosoma brucei possesses a large family of transmembrane receptor-like adenylate cyclases (RACs), primarily located to the flagellar surface and involved in sensing of the extracellular environment. RACs exhibit a conserved topology characterized by a large N-terminal extracellular moiety harbouring two Venus Flytrap (VFT) bilobate structures separated from an intracellular catalytic domain by a single transmembrane helix. RAC activation, which typically occurs under mild acid stress, requires the dimerization of the intracellular catalytic domain. The occurrence of VFT domains in the RAC's extracellular moiety suggests their potential responsiveness to extracellular ligands in the absence of stress, although no such ligands have been identified so far. Herein we report the biophysical characterization of the membrane-proximal VFT2 domain of a bloodstream form-specific RAC called ESAG4, whose ectodomain 3D structure is completely unknown. The paper describes an AlphaFold2-based optimisation of the expression construct, enabling facile and high-yield recombinant production and purification of the target protein. Through an interdisciplinary approach combining various biophysical methods, we demonstrate that the optimised VFT2 domain obtained by recombination is properly folded and behaves as a monomer in solution. The latter suggests a ligand-binding capacity independent of dimerization, unlike typical mammalian VFT receptors, as guanylate cyclase. In silico VFT2 genomic analyses shows divergence among cyclase isoforms, hinting at ligand specificity. Taken together this improved procedure enabling facile and high-yield recombinant production and purification of the target protein could benefit researchers studying trypanosomal RAC VFT domains but also any trypanosome domain with poorly defined boundaries. Additionally, our findings support the stable monomeric VFT2 domain as a useful tool for future structural investigations and ligand screening.
      Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
      (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
    • Contributed Indexing:
      Keywords: Adenylate cyclase; Ligand; Small angle X-ray scattering; Thermal shift assay; Trypanosoma brucei; Venus Flytrap domain
    • Accession Number:
      EC 4.6.1.1 (Adenylyl Cyclases)
      0 (Protozoan Proteins)
      0 (Recombinant Proteins)
    • Publication Date:
      Date Created: 20241024 Date Completed: 20241201 Latest Revision: 20241222
    • Publication Date:
      20241222
    • Accession Number:
      10.1016/j.molbiopara.2024.111653
    • Accession Number:
      39447762