Affiliation:
1. Hefei National Research Center for Physical Sciences at the Microscale, MOE Key Laboratory for Membraneless Organelles & Cellular Dynamics, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, Biomedical Sciences and Health Laboratory of Anhui Province, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, China
2. Department of Neurology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, China
Abstract
The ankyrin repeat-rich membrane spanning (ARMS), a transmembrane neuronal scaffold protein, plays a fundamental role in neuronal physiology, including neuronal development, polarity, differentiation, survival and angiogenesis, through interactions with diverse partners. Previous studies have shown that the ARMS negatively regulates brain-derived neurotrophic factor (BDNF) secretion by interacting with Synaptotagmin-4 (Syt4), thereby affecting neurogenesis and the development and function of the nervous system. However, the molecular mechanisms of the ARMS/Syt4 complex assembly remain unclear. Here, we confirmed that the ARMS directly interacts with Syt4 through its N-terminal ankyrin repeats 1–8. Unexpectedly, both the C2A and C2B domains of Syt4 are necessary for binding with the ARMS. We then combined the predicted complex structural models from AlphaFold2 with systematic biochemical analyses using point mutagenesis to underline the molecular basis of ARMS/Syt4 complex formation and to identify two conserved residues, E15 and W72, of the ARMS, as essential residues mediating the assembly of the complex. Furthermore, we showed that ARMS proteins are unable to interact with Syt1 or Syt3, indicating that the interaction between ARMS and Syt4 is specific. Taken together, the findings from this study provide biochemical details on the interaction between the ARMS and Syt4, thereby offering a biochemical basis for the further understanding of the potential mechanisms and functional implications of the ARMS/Syt4 complex formation, especially with regard to the modulation of BDNF secretion and associated neuropathies.
Funder
National Natural Science Foundation of China
Center for Advanced Interdisciplinary Science and Biomedicine of IHM
USTC Research Funds of the Double First-Class Initiative
Fundamental Research Funds for the Central Universities
Strategic Priority Research Program of the Chinese Academy of Sciences
Subject
Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis