Neuronal Scaffold Protein ARMS Interacts with Synaptotagmin-4 C2AB through the Ankyrin Repeat Domain with an Unexpected Mode

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.