Poster Presentation 49th Lorne Conference on Protein Structure and Function 2024

Structural insights into how the membrane protein TMEM120A interacts with Mechanosenstive Ion Channel Inhibitors (#207)

Mayada Mazhar 1 2 , Hamish Brown 1 3 , Michael W. Parker 1 2 , Isabelle Rouiller 1 2
  1. Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, VIC, Australia
  2. ARC Centre for Cryo-electron Microscopy of Membrane Proteins, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, VIC, Australia
  3. Ian Holmes Imaging Centre, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, VIC, Australia

Human TMEM120A, or TACAN, is a membrane protein proposed to be a mechanosensitive ion channel (MSC) [1]. However, several electrophysiology studies showed it does not act as MSC [2,3,4]. A recent study showed TACAN may constitute a fundamentally unique type of MSC [5]. Molecular dynamic simulations and electrophysiology experiments indicated that M207 in wild-type TACAN appears to function as a gate to obstruct the hypothesised ion conduction channel [5]. However, there is not yet any published structural data showing if and how TACAN interacts with MSC inhibitors such as GSMTX4 and GdCl3.

One of our study aims is to reveal the role of TACAN, whether it's an MSC or MSC regulator molecule, by determining the structure of TACAN with MSC inhibitors. We will achieve this aim by biophysical binding assays, electrophysiology studies, and cryo-EM studies on TACAN with both Inhibitors. Here, we present biophysical binding assays of human TACAN with GSMTX4 and GdCl3. We performed autodocking of the TACAN monomer with GSMTX4 using cluspro2 software [6]. Docking showed binding of GSMTX4 near the proposed ion channel [5]. Then, we expressed Flag-tagged human TACAN in HEK293T SF 17 cells grown in suspension and purified the protein using anti-FLAG M2 column affinity chromatography followed by Size-Exclusion Chromatography. NanoDSF showed that GSMTX4 and GdCl3 stabilised the protein. Circular Dichroism data showed a slight change in the conformation and secondary structure of the protein with both inhibitors at higher doses (30 uM GSMTX4 and 100 uM GdCl3). Mass photometry showed no aggregation of the protein at low doses of inhibitors (5 uM GSMTX4 and 30 uM GdCl3). However, a high dose of 30 uM GSMTX4 showed protein aggregation. Our data support protein binding with both inhibitors at low dose with associated changes in protein conformation. Our next step is to visualise how the inhibitors bind to TACAN using cryo-electron microscopy.

  1. Beaulieu-Laroche, L., et al. (2020). "TACAN Is an Ion Channel Involved in Sensing Mechanical Pain." Cell 180(5): 956-967.e917.
  2. Niu, Y., et al. (2021). "Analysis of the mechanosensory channel functionality of TACAN." Elife 10.
  3. Rong, Y., et al. (2021). "TMEM120A contains a specific coenzyme A-binding site and might not mediate poking- or stretch-induced channel activities in cells." Elife 10.
  4. Xue, J., et al. (2021). "TMEM120A is a coenzyme A-binding membrane protein with structural similarities to ELOVL fatty acid elongase." Elife 10.
  5. Chen, X., et al. (2022). "Cryo-EM structure of the human TACAN in a closed state." Cell Rep 38(9): 110445.
  6. Kozakov, Dima et al. “The ClusPro web server for protein-protein docking.” Nature protocols vol. 12,2 (2017): 255-278. doi:10.1038/nprot.2016.169